FGF7/<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) regulates the differentiation and development of the prostate epithelium, while over-expression of FGF8 and FGF1 are implicated in carcinogenesis of the prostate. We tested the hypothesis that different members of the FGF family function through different signalling molecules. In prostate DU145 cells, both FGF1 and FGF<em>2</em> activated ERK1/<em>2</em> potently and p38 moderately. KGF was however most efficient in inducing p38 activities but had no effect on ERK1/<em>2</em> function. JNK and STAT activities were not induced by FGFs in prostate cells. In vitro expression of the transcription <em>factors</em> Elk-1 and MEF<em>2</em>A (substrates for ERK1/<em>2</em> and p38, respectively) for functional quantification, confirmed the pattern of FGF-induced MAPK activations in COS-7 cells. Furthermore, KGF was more efficient than FGF1 and FGF<em>2</em> in inducing actin stress fibres, and the specific p38 inhibitor SB<em>2</em>0<em>2</em>190 completely abolished this in a dose-dependent manner. The MEK1/<em>2</em> inhibitor, U01<em>2</em>6, had no effect on FGF-induced stress fibre formation. This study demonstrates the selective activation of MAPK family members by FGFs resulting in activation of transcription <em>factors</em> and stress fibre formation. As multiple FGFs are over-expressed in human prostate cancer, characterization of the distinct signalling pathway by FGFs may reveal new specific targets for therapy.

Recent evidence suggests that the phosphocholine-derived lipid mediator platelet-activating <em>factor</em> (PAF) is involved in <em>keratinocyte</em> function and cutaneous inflammation. PAF is found in various inflammatory skin diseases, and intradermal injection of PAF directly results in cutaneous inflammation. <em>Keratinocytes</em> also synthesize PAF and related 1-acyl species in response to ionophores, cytokines and <em>growth</em> <em>factors</em>, and in response to activation of the epidermal PAF receptor. Since <em>keratinocytes</em> are routinely exposed to potential damage by thermal or oxidative stressors with resultant induction of cutaneous inflammation, the objective of these studies was to assess whether exogenous thermal or oxidative damage can induce the production of PAF and related 1-acyl species. Cells of the immortalized human <em>keratinocyte</em> cell line HaCaT were subjected to acute heat or cold, or treatment with the pro-oxidant lipid tertiary butyl hydroperoxide, and PAF and 1-palmitoyl-<em>2</em>-acetyl-GPC were measured by gas chromatography/mass spectrometry. We report that these diverse toxic stimuli resulted in the accumulation of these biologically active lipids. These studies suggest that the PAF system is involved in the inflammatory response seen following acute epidermal damage.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) are known mitogens for normal alveolar Type <em>2</em> cells in vitro and in vivo. We wished to determine whether these two <em>growth</em> <em>factors</em> are involved in lung repair after epithelial cell necrosis by determining the levels of each <em>factor</em> in lung lavage fluid collected serially after bleomycin-induced injury, and how these values relate specifically to proliferation of bronchiolar and alveolar epithelial cells. Rats received an intratracheal injection of 1 unit bleomycin in 0.5 ml water and were killed at intervals up to 4 weeks with 1 muCi/g tritiated thymidine injected 1 hour before death. Early necrosis of bronchiolar epithelial (BR) cells and Type 1 alveolar epithelium was followed by an increase in inflammatory cell numbers and high protein levels in bronchoalveolar lavage (BAL) fluids. In addition, the levels of KGF and HGF, measured by enzyme-linked immunosorbent assay in BAL, increased as early as 3 days and peaked at 7-14 days, when KGF was measured at 160 pg/ml (n = 50) and HGF reached 460 pg/ml (n = 40). Both values dropped sharply after <em>2</em> weeks. Epithelial cell proliferation was quantitated as percentage of labeled cells in autoradiographs of methacrylate sections. Labeling of BR cells predominated in the first week and peaked at 7% at 3 days. Type <em>2</em> cell proliferation was delayed somewhat but occurred in 3 to 10 days with a peak of 7% labeled cells at 1 week. The results demonstrate that both HGF and KGF are present in the lung in greatly increased amounts soon after bleomycin-induced epithelial cell necrosis. These high levels are associated with both BR and alveolar epithelial cell proliferation.

BACKGROUND

Vitiligo is an acquired pigmentary disorder characterized by depigmentation of skin and hair. As the pathogenesis of this disease is still obscure, the treatment of vitiligo has generally been unsatisfactory and often disappointing. Topical tacrolimus (FK506) ointment has recently been added to the armamentarium against this pigmentary disorder. Despite its clinical efficacy, the underlying mechanisms of how topical tacrolimus induces repigmentation in vitiligo have rarely been investigated. As tacrolimus ointment is applied directly to the skin, its impact on keratinocytes (KCs) requires thorough investigation.

OBJECTIVE

To investigate the effects of FK506 on melanocyte (MC) and melanoblast (MB) growth via KCs.

METHODS

Cultured MCs and MBs were treated with supernatant of KC cultures conditioned with various concentrations of FK506. The impact of supernatant on MCs and MBs was assessed in terms of its effect on MC/MB proliferation, melanin formation and cell migration. The activities of matrix metalloproteinase (MMP)-2 and MMP-9, known for their influence on cell migration, were evaluated. The concentrations of MC/MB growth factors in the KC supernatant were also determined.

RESULTS

Results demonstrated that proliferation of both MCs and MBs was significantly enhanced by FK506-treated KC supernatant. In addition, the concentration of stem cell factor in KC supernatant increased dose-dependently with FK506 treatment. The supernatant from FK506-treated KC culture showed a significant increase in MMP-9 activity.

CONCLUSIONS

Our study provides in vitro evidence demonstrating that direct interaction between FK506 and KCs creates a favourable milieu for MC growth and migration. Furthermore, our findings provide a possible mechanism explaining how tacrolimus ointment induces repigmentation in patients with vitiligo.

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:<em>2</em>0 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as <em>growth</em> <em>factors</em>. DSC analysis revealed the characteristic melting point of PCL at around 60 degrees C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of <em>2</em> x 10(5)/cm(<em>2</em>) on 1:<em>2</em>0 collagen:PCL materials at day 8 with cell numbers declining thereafter. <em>Keratinocyte</em> <em>growth</em> rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6 x 10(4)/cm(<em>2</em>) at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for <em>growth</em> of fibroblasts and <em>keratinocytes</em> and may find utility for skin repair.

There is increasing evidence that G-protein-coupled receptors cross-talk with <em>growth</em> <em>factor</em> receptor-mediated signal transduction in a variety of cell types. We have investigated mechanisms by which the activation of beta-adrenergic receptors, classically GTP-binding proteins coupled receptors, influence the migration of cultured human <em>keratinocytes</em>. We found that iso-proterenol, a beta-adrenergic receptor-selective agonist, inhibited cell migration stimulated by either epidermal <em>growth</em> <em>factor</em>, or extracellular Ca<em>2</em>+ in a concentration-dependent manner. This was prevented by pretreatment of the cells with the beta-adrenergic receptor-selective antagonist timolol. Interestingly, isoproterenol, at a concentration of 1 nm, did not measurably increase intracellular cyclic adenosine monophosphate concentrations yet inhibited cell migration by 50%. To test further if isoproterenol's actions were mediated via activation of adenylyl cyclase, two inhibitors of its activity, <em>2</em>'5'-dideoxyadenosine and SQ<em>2</em><em>2</em>536, were used. Both compounds significantly diminished iso-proterenol-induced increases in intracellular cyclic adenosine monophosphate concentrations but did not attenuate isoproterenol-induced inhibition of cell migration. Also, forskolin (1 microm) markedly increased intracellular cyclic adenosine monophosphate concentrations but did not significantly inhibit cell migration. As mitogen-activated protein kinases are known to signal <em>growth</em> <em>factor</em>-stimulated cell migration, we examined whether beta-adrenergic receptor-mediated inhibition of <em>keratinocyte</em> migration might occur via inactivation of mitogen-activated protein kinases. We found that isoproterenol inhibited phosphorylation of extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration-dependent manner but had no effect on the phosphorylation of the stress mitogen-activated protein kinases c-jun N-terminal kinase and stress-activated protein kinase-<em>2</em>. Neither forskolin nor a membrane permeable cyclic adenosine monophosphate analog inhibited phosphorylation of any of these mitogen-activated protein kinases. These findings suggest that beta-adrenergic receptor-induced inhibition of <em>keratinocyte</em> migration is mediated through inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase signaling in a cyclic adenosine monophosphate-independent manner.

Ultraviolet (UV) irradiation causes photoageing through induction of matrix-degrading metalloproteinases (MMP), which are upregulated by activator protein-1 (AP-1) (Jun/Fos). The c-Jun kinase activity proves to be critically important in the regulation of AP-1 activity. Our previous studies showed that UV irradiation activates epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and cytokine receptors leading to the activation of c-Jun kinase in cultured human skin <em>keratinocytes</em> in vitro and in human skin in vivo. However, the mechanism of UV-induced cell surface receptor activation and the crosstalk among <em>growth</em> <em>factor</em> receptor and cytokine receptors were not fully investigated. This study showed that UV (30 mJ/cm(<em>2</em>))-induced EGFR tyrosine phosphorylation in a manner similar to EGF (100 ng/ml), or IL-1beta (10 ng/ml) in cultured human <em>keratinocytes</em>. In all cases, EGFR tyrosine phosphorylation was completely inhibited by pretreatment of PD153035 (100 nM, 1 h). Also observed was that UV induced autophosphorylation of interleukin 1 receptor associated kinase (IRAK) in a manner analogous to IL-1beta or EGF. In both UV and EGF cases, the phosphorylation of IRAK was inhibited by pretreatment of PD153035. However, IL-1beta-induced IRAK activation was not affected by PD153035. In vitro kinase assay using GST-c-Jun as a substrate revealed that pretreatment of PD153035 completely inhibited UV- and IL-1-induced c-Jun kinase activity in cultured <em>keratinocytes</em>. Taken together, the above data suggest that EGFR plays dominant role in the crosstalk among <em>growth</em> <em>factor</em> receptor and cytokine receptors leading to the activation of c-Jun kinase upon UV irradiation, and that EGFR could be one of the targets for clinical and cosmetical prevention of UV-induced skin aging.

The oral epithelial barrier separates the host from the environment and provides the first line of defense against pathogens, exogenous substances and mechanical stress. It consists of underlying connective tissue and a stratified keratinized epithelium with a basement membrane, whose cells undergo terminal differentiation resulting in the formation of a mechanically resistant surface. Gingival <em>keratinocytes</em> are connected by various transmembrane proteins, such as tight junctions, adherens junctions and gap junctions, each of which has a specialized structure and specific functions. Periodontal pathogens are able to induce inflammatory responses that lead to attachment loss and periodontal destruction. A number of studies have demonstrated that the characteristics of pathogenic oral bacteria influence the expression and structural integrity of different cell-cell junctions. Tissue destruction can be mediated by host cells following stimulation with cytokines and bacterial products. <em>Keratinocytes</em>, the main cell type in gingival epithelial tissues, express a variety of proinflammatory cytokines and chemokines, including interleukin-1alpha, interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis <em>factor</em>-alpha. Furthermore, the inflammatory mediators that may be secreted by oral <em>keratinocytes</em> are vascular endothelial <em>growth</em> <em>factor</em>, prostaglandin E<em>2</em> , interleukin-1 receptor antagonist and chemokine (C-C motif) ligand <em>2</em>. The protein family of matrix metalloproteinases is able to degrade all types of extracellular matrix protein, and can process a number of bioactive molecules. Matrix metalloproteinase activities under inflammatory conditions are mostly deregulated and often increased, and those mainly relevant in periodontal disease are matrix metalloproteinases 1, <em>2</em>, 3, 8, 9, 13 and <em>2</em>4. Viral infection may also influence the epithelial barrier. Studies show that the expression of HIV proteins in the mucosal epithelium is correlated with the disruption of epithelial tight junctions, suggesting a possible enhancement of human papilloma virus infection by HIV-associated disruption of tight junctions. Altered expression of matrix metalloproteinases was demonstrated in <em>keratinocytes</em> transformed with human papilloma virus-16 or papilloma virus-18,. To summarize, the oral epithelium is able to react to a variety of exogenous, possibly noxious influences.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) deregulates epithelial homeostasis through the modulation of receptor tyrosine kinases and their signaling. Accordingly, the fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>b (FGFR<em>2</em>b/KGFR), epithelial splicing transcript variant of the FGFR<em>2</em>, is down-modulated by the viral protein expression, leading to impairment of <em>keratinocyte</em> differentiation. Here, we report that, in cell models of transfected human <em>keratinocytes</em> as well as in cervical epithelial cells containing episomal HPV16, the down-regulation of FGFR<em>2</em>b induced by 16E5 is associated with the aberrant expression of the mesenchymal FGFR<em>2</em>c isoform as a consequence of splicing switch: in fact, quantitative RT-PCR analysis showed that this molecular event is transcriptionally regulated by the epithelial splicing regulatory proteins 1 and <em>2</em> (ESRP1 and ESRP<em>2</em>) and is able to produce effects synergistic with those caused by TGFβ treatment. Immunofluorescence analysis revealed that this altered FGFR<em>2</em> splicing leads to changes in the specificity for the ligands FGFs and in the cellular response, triggering epithelial-mesenchymal transition (EMT). Through 16E5 or FGFR<em>2</em> silencing as well as inhibition of FGFR<em>2</em> activity we demonstrated the direct role of the viral protein in the receptor isoform switching and EMT, suggesting that these early molecular events during HPV infection might represent additional mechanisms driving cervical transformation and tumor progression.

OBJECTIVE

The purposes of this study were: to review studies of growth factors in cutaneous wounds of animals with diabetes to identify those factors with altered gene expression and content compared with nondiabetic animals; and to explore which deficiencies of growth factors in diabetic wounds may or may not be improved by laser irradiation.

BACKGROUND

Wound healing is compromised in diabetes. Decreased production and/or increased destruction of growth factors may be responsible. Laser irradiation can increase the gene expression and release of certain growth factors by cells.

METHODS

Research articles investigating growth factor expression in wounds of nondiabetic and diabetic mice and rats published through September 2011 were retrieved from library sources, PubMed databases, reference lists of articles, and searches of relevant journals.

RESULTS

Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), keratinocyte growth factor (KGF), fibroblast growth factor 1 (FGF-1), FGF-2, insulin-like growth factor 1 (IGF-1), IGF-2, transforming growth factor beta (TGF-β), and nerve growth factor (NGF) had decreased gene expression and content in early phases of healing for diabetic wounds. Gene expression of KGF, IGF-1, and IGF-2 was delayed, whereas that of FGF-1 and FGF-2 occurred earlier, in diabetic compared with nondiabetic wounds.

CONCLUSIONS

Growth factor administration combined with laser irradiation may provide an effective therapy to maximize healing of diabetic wounds.

Plasminogen activator inhibitor type-1 (PAI-1) is the major negative regulator of the plasmin-dependent pericellular proteolytic cascade. PAI-1 gene expression is normally <em>growth</em> state regulated but frequently elevated in chronic fibroproliferative and neoplastic diseases affecting both stromal restructuring and cellular migratory activities. Kinetic modeling of cell cycle transit in synchronized human <em>keratinocytes</em> (HaCaT cells) indicated that PAI-1 transcription occurred early after serum stimulation of quiescent (G0) cells and prior to entry into a cycling G1 condition. PAI-1 repression (in G0) was associated with upstream stimulatory <em>factor</em>-1 (USF-1) occupancy of two consensus E box motifs (5'-CACGTG-3') at the PE1 and PE<em>2</em> domains in the PF1 region (nucleotides -794 to -53<em>2</em>) of the PAI-1 promoter. Chromatin immunoprecipitation (ChIP) analysis established that the PE1 and PE<em>2</em> site E boxes were occupied by USF-1 in quiescent cells and by USF-<em>2</em> in serum-activated, PAI-1-expressing <em>keratinocytes</em>. This reciprocal and <em>growth</em> state-dependent residence of USF family members (USF-1 vs. USF-<em>2</em>) at PE1/PE<em>2</em> region chromatin characterized the G0 ->> G1 transition period and the transcriptional status of the PAI-1 gene. A consensus E box motif was required for USF/E box interactions, as a CG ->> AT substitution at the two central nucleotides inhibited formation of USF/probe complexes. The 5' flanking sites (AAT or AGAC) in the PE<em>2</em> segment were not necessary for USF binding. USF recognition of the PE1/PE<em>2</em> region E box sites required phosphorylation with several potential involved residues, including T153, maping to the USF-specific region (USR). A T153A substitution in USF-1 did not repress serum-induced PAI-1 expression whereas the T153D mutant was an effective suppressor. As anticipated from the ChIP results, transfection of wild-type USF-<em>2</em> failed to inhibit PAI-1 induction. Collectively, these data suggest that USF family members are important regulators of PAI-1 gene control during serum-stimulated recruitment of quiescent human epithelial cells into the <em>growth</em> cycle.

Squamous cell carcinoma (SCC) is an invasive malignancy of epidermal <em>keratinocytes</em>. Surgical excision is currently the main treatment; however, this can cause scarring and disfigurement. There is accordingly, an acute need for alternative strategies to treat SCC. The transcription <em>factor</em> c-Jun is expressed in human SCC and another common form of invasive skin cancer, basal cell carcinoma together with the mitogenic marker-proliferating cell nuclear antigen. Here, we have employed DNAzymes (catalytic DNA molecules) targeting c-Jun (Dz13) to inhibit c-Jun expression in SCC cells. Dz13 inhibits SCC proliferation and suppresses solid SCC tumor <em>growth</em> and tumor angiogenesis in severe combined immunodeficient mice. We further demonstrate that Dz13 inhibits c-Jun, together with matrix metalloproteinase (MMP)-<em>2</em> and MMP-9 expression in the tumors, consistent with DNAzyme inhibition of MMP-<em>2</em> and MMP-9 gelatinolytic activity by zymography. Dz13 also suppressed the expression of vascular endothelial <em>growth</em> <em>factor</em> and fibroblast <em>growth</em> <em>factor</em>-<em>2</em> in the tumors. These findings demonstrate that c-Jun regulates SCC <em>growth</em> and suggest that DNAzymes targeting this transcription <em>factor</em> may potentially be useful as inhibitors of cutaneous carcinoma.

The mechanisms of pulmonary repair in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are poorly known. Hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) are key <em>factors</em> involved in alveolar epithelial repair, present in the bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS. The role of BALF mediators in their production remains to be determined. We evaluated the overall effect of BALF from 5<em>2</em> patients (<em>2</em>7 ventilated patients with ALI/ARDS, 10 ventilated patients without ALI, and 15 nonventilated control patients) on HGF and KGF synthesis by lung fibroblasts. Fibroblasts were cultured in the presence of BALF. HGF and KGF protein secretion was measured using ELISA, and mRNA expression was evaluated using quantitative real-time RT-PCR. Only BALF from ALI/ARDS patients upregulated both HGF and KGF mRNA expression and protein synthesis (+<em>2</em>71 and +146% for HGF and KGF, respectively). BALF-induced HGF synthesis from ALI/ARDS patients was higher than that from ventilated patients without ALI (P < 0.05). HGF secretion was correlated with BALF IL-1beta levels (rho = 0.6<em>2</em>, P < 0.001) and BALF IL-1beta/IL-1 receptor antagonist ratio (rho = 0.54, P < 0.007) in the ALI/ARDS group. An anti-IL-1beta antibody partially (>50%) inhibited the BALF-induced HGF and PGE(<em>2</em>) secretion, whereas NS-398, a specific cyclooxygenase-<em>2</em> (COX-<em>2</em>) inhibitor, completely inhibited it. Anti-IL-1beta antibodies as well as NS-398 reversed the COX-<em>2</em> upregulation induced by BALF. Therefore, IL-1beta is a main BALF mediator involved in HGF secretion, which is mediated through a PGE(<em>2</em>)/COX-<em>2</em>-dependent mechanism. BALF mediators may participate in vivo in the production of HGF and KGF by lung fibroblasts during ALI/ARDS.

Viperid snakebite envenomation induces blistering and dermonecrosis. The pathological alterations induced by a snake venom metalloproteinase in the skin were investigated in a mouse ear model. Metalloproteinase BaP1, from Bothrops asper, induced rapid edema, hemorrhage, and blistering; the latter two effects were abrogated by preincubation with the metalloproteinase inhibitor batimastat. Neutrophils did not play a role in the pathology, as depletion of these cells resulted in a similar histological picture. Blisters are likely to result from the direct proteolytic activity of BaP1 of proteins at the dermal-epidermal junction, probably at the lamina lucida, as revealed by immunostaining for type IV collagen and laminin. Widespread apoptosis of <em>keratinocytes</em> was detected by the TUNEL assay, whereas no apoptosis of capillary endothelial cells was observed. BaP1 induced a drastic reduction in the microvessel density, revealed by immunostaining for the endothelial marker vascular endothelial <em>growth</em> <em>factor</em> receptor-<em>2</em>. This was followed by a rapid angiogenic response, leading to a partial revascularization. Skin damage was followed by inflammation and granulation tissue formation. Then, a successful re-epithelization process occurred, and the skin of the ear regained its normal structure by <em>2</em> weeks. Venom metalloproteinase-induced skin damage reproduces the pathological changes described in snakebitten patients.

MMP-8 (collagenase-<em>2</em>) is the most effective collagenase to initiate type I collagen degradation. Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP-8 in squamous cell carcinoma (SCC) of the head and neck in vivo and in vitro. Most of the recently established head and neck carcinoma cell lines (<em>2</em><em>2</em>/<em>2</em>5), corresponding tumour (5/7) and dermal (<em>2</em>/<em>2</em>) fibroblasts, commercial tongue carcinoma (HSC-3 and SCC-<em>2</em>5), and transformed <em>keratinocyte</em> cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP-8 mRNA analysed by the PCR method. Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal fibroblasts. The expression of immunoreactive MMP-8 protein was reduced 30% by transforming <em>growth</em> <em>factor</em> beta-1 (TGF-beta1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up-regulated <em>2</em>- and <em>2</em>.5-fold after 10 nM and 100 nM phorbol 1<em>2</em>-myristate 13 acetate (PMA), respectively. Immunohistological staining localized MMP-8 protein in a few malignant invading tumour cell islands, certain fibroblasts, polymorphonuclear neutrophils (PMNs), and plasma cells. In situ hybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed. It is concluded that tissue from head and neck carcinomas can express MMP-8 both in vivo and in vitro. Since the amount of MMP-8 in carcinoma and stromal cells is rather low, MMP-8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma.

Akt signaling is involved in tumorigenesis via a number of different mechanisms that result in increased proliferation and decreased apoptosis. Previous data have demonstrated that Akt-mediated signaling is functionally involved in <em>keratinocyte</em> transformation. This work investigates the involvement of angiogenesis as a mediator of tumorigenesis in Akt-transformed <em>keratinocytes</em>. Tumors produced by subcutaneous injection of the latter showed increased angiogenic profiles associated with increased vascular endothelial <em>growth</em> <em>factor</em> (VEGF) protein levels. However, in contrast to v-ras(Ha)-transformed <em>keratinocytes</em>, VEGF mRNA levels were not increased. The induction of VEGF protein by Akt is associated with increased phosphorylation and thus activation of p70S6K and eIF4E-binding protein 1, leading to increased VEGF translation. In addition, we observed increased metaloproteinases <em>2</em> and 9 expression, but not thrombospondin 1, in tumors derived from Akt-transformed <em>keratinocytes</em>. Collectively, these results demonstrate that Akt is an important mediator of angiogenesis in malignant <em>keratinocytes</em> through a post-transcriptional mechanism.

The peripheral nervous system, in addition to its sensory and motor functions, can induce a local inflammatory response known as neurogenic inflammation. This phenomenon plays a critical role in several inflammatory diseases, e.g., asthma, atopy, rheumatoid arthritis, psoriasis, and ulcerative colitis. Neurogenic inflammation and the role of nerve <em>growth</em> <em>factor</em> (NGF) have been extensively studied in psoriasis. There are increased levels of NGF in the <em>keratinocytes</em> and upregulation of NGF receptor (NGF-R) in the cutaneous nerves of psoriatic plaques. NGF can influence all the salient pathologic events noticed in psoriasis such as proliferation of <em>keratinocytes</em>, angiogenesis, T cell activation, expression of adhesion molecules, proliferation of cutaneous nerves, and upregulation of neuropeptides. In this double-blinded, placebo-controlled study, we addressed the role of NGF/NGF-R in psoriasis in an in vivo system using the severe combined immunodeficient (SCID) mouse-human skin model of psoriasis. The transplanted psoriatic plaques on the SCID mice (n=1<em>2</em>) were treated with K<em>2</em>5<em>2</em>a, a high-affinity NGF receptor blocker. Psoriasis significantly improved following <em>2</em> wk of therapy. The length of the rete pegs changed from 308.57+/-98.7<em>2</em> to 164.64+/-46.78 microm (p<0.01, Student's t test). A similar improvement of psoriasis was observed by directly inhibiting NGF with NGF-neutralizing antibody. NGF-neutralizing antibody in normal saline at 10 ng (n=4) and <em>2</em>0 ng (n=4) per kilogram of body weight doses were used. Both doses of NGF-neutralizing antibody reduced rete peg lengths significantly, e.g., from <em>2</em>98.5+/-4<em>2</em>.69 to 150.5<em>2</em>+/-3<em>2</em>.93 microm (p<0.05, Student's t test). This study provides evidence for the role of NGF and its high-affinity receptor in the pathogenesis of psoriasis and insights to develop novel therapeutic modalities.

Apoptosis plays an important role in eliminating dysfunctional damaged cells. For skin, the best characterized injurious environmental agent is ultraviolet (UV) irradiation. Most of the damaging UV irradiation is absorbed in the epidermis and leads to apoptosis of <em>keratinocytes</em>. However, epidermal melanocytes appear to be protected from UV-induced apoptosis. We now report that in pure cultures melanocytic cells undergo characteristic apoptosis after physiologic UV exposures. However, nerve <em>growth</em> <em>factor</em> (NGF) supplementation protects them from this programmed cell death. Furthermore, we show that NGF protects melanocytic cells from UV-induced apoptosis by upregulating BCL-<em>2</em> protein in these cells and that prior downregulation of BCL-<em>2</em> abrogates the NGF protective effect on melanocytes. Our data suggest that NGF, known to be constitutively produced by epidermal <em>keratinocytes</em> and induced in these cells after UV irradiation, may preserve the population of cutaneous melanocytes that would otherwise be depleted by casual sun exposure.

OBJECTIVE

Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satis<em>factor</em>y agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast <em>growth</em> <em>factor</em> <em>2</em> (FGF-P) was synthesized and its mitigation effect on AGS was examined.

METHODS

A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.<em>2</em> ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis <em>factor</em> (TNF)-alpha were evaluated.

RESULTS

Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses.

CONCLUSIONS

The study data support pursuing FGF-P as a mitigator for AGS.

OBJECTIVE

Radiosensitivity of stem cells is a matter of debate. For mouse somatic stem cells, both radiosensitive and radioresistant stem cells have been described. By contrast, the response of human stem cells to radiation has been poorly studied. As epidermis is a radiosensitive tissue, we evaluated in the present work the radiosensitivity of cell populations enriched for epithelial stem cells of human epidermis.

METHODS

The total <em>keratinocyte</em> population was enzymatically isolated from normal human skin. We used flow cytometry and antibodies against cell surface markers to isolate basal cell populations from human foreskin. Cell survival was measured after a dose of <em>2</em>Gy with the XTT assay at 7<em>2</em>h after exposure and with a clonogenic assay at <em>2</em> weeks. Transcriptome analysis using oligonucleotide microarrays was performed to assess the genomic cell responses to radiation.

RESULTS

Cell sorting based on two membrane proteins, alpha6 integrin and the transferrin receptor CD71, allowed isolation of <em>keratinocyte</em> populations enriched for the two types of cells found in the basal layer of epidermis: stem cells and progenitors. Both the XTT assay and the clonogenic assay showed that the stem cells were radioresistant whereas the progenitors were radiosensitive. We made the hypothesis that upstream DNA damage signalling might be different in the stem cells and used microarray technology to test this hypothesis. The stem cells exhibited a much more reduced gene response to a dose of <em>2</em>Gy than the progenitors, as we found that 6% of the spotted genes were regulated in the stem cells and <em>2</em>0% in the progenitors. Using Ingenuity Pathway Analysis software, we found that radiation exposure induced very specific pathways in the stem cells. The most striking responses were the repression of a network of genes involved in apoptosis and the induction of a network of cytokines and growth factors.

CONCLUSIONS

These results show for the first time that keratinocyte populations enriched for stem cells from human epidermis are radioresistant. Based on both repressed and induced genes, we found that the major response of the irradiated stem cell population was the regulation of genes functionally related to cell death, cell survival and apoptosis.

Inflammatory malignant fibrous histiocytomas (IMFH) are rare tumors and are frequently associated with leukocytosis. In rare cases, leukemoid reactions were attributed to tumor production of unidentified hematopoietic <em>factors</em>. In this study, we used immunohistochemical techniques to show cytokine immunoreactivity in the malignant cells of two cases of IMFH presenting with leukemoid reactions and compared them with two malignant fibrous histocytomas, noninflammatory type. All four tumors stained positively for stem cell <em>factor</em> (SCF), granulocyte colony-stimulating <em>factor</em> (G-CSF), interleukin-<em>2</em> (IL-<em>2</em>), IL-4, IL-5, interferon-alpha (IFN-alpha), and insulin-like <em>growth</em> <em>factor</em>-I. Other cytokines detected only in the two IMFH included IL-6, IL-7, IL-8, IFN-gamma, and <em>keratinocyte</em> <em>growth</em> <em>factor</em>. Granulocyte-macrophage-CSF, IL-3, and transforming <em>growth</em> <em>factor</em>-beta staining was present in one of the two IMFH tumors and was not present in the noninflammatory tumors. The immunohistochemical staining was localized to the malignant cells, suggesting deregulated cytokine expression consistent with their monocytic/histocytic origin. Expression of certain cytokines in the IMFH may account for the local inflammatory infiltrate, tumor fibrosis, and the aggressive nature of the malignant cells. We also detected elevated serum levels of SCF, G-CSF, IL-6, and tumor necrosis <em>factor</em> in one or both of the IMFH patients. These latter observations may explain the bone marrow hypercellularity and other paraneoplastic symptoms, including fever, malaise, and weight loss, observed in both patients. Different cytokines present in the two IMFH tumors appear to be responsible for the eosinophilic leukemoid reaction observed in one case and for the granulocytic leukemoid reaction observed in the other patient. They may also be responsible for expansion of the tumor-cell population, fibroblast proliferation, and enhanced secretion of extracellular collagen.

Wound healing requires re-epithelialization from the wound margin through <em>keratinocyte</em> proliferation and migration, and some <em>growth</em> <em>factors</em> are known to influence this process. In the present study, we found that the co-treatment with hepatocyte <em>growth</em> <em>factor</em> (HGF) and TGF-beta1 resulted in enhanced migration of HaCaT cells compared with either <em>growth</em> <em>factor</em> alone, and that N-acetylcysteine, an antioxidant agent, was the most effective among several inhibitors tested, suggesting the involvement of reactive oxygen species (ROS). Fluorescence-activated cell sorter analysis using <em>2</em>,7-dichlorofluorescein diacetate (DCF-DA) dye showed an early (30 min) as well as a late (<em>2</em>4 h) increase of ROS after scratch, and the increase was more prominent with the <em>growth</em> <em>factor</em> treatment. Diphenyliodonium (DPI), a potent inhibitor of NADPH oxidase, abolished the increase of ROS at 30 min, followed by the inhibition of migration, but not the late time event. More precisely, gene knockdown by shRNA for either Nox-1 or Nox-4 isozyme of gp91phox subunit of NADPH oxidase abolished both the early time ROS production and migration. However, HaCaT cell migration was not enhanced by treatment with H((<em>2</em>))O((<em>2</em>)). Collectively, co-treatment with HGF and TGF-beta1 enhances <em>keratinocyte</em> migration, accompanied with ROS generation through NADPH oxidase, involving Nox-1 and Nox-4 isozymes.

The regulation of epidermal <em>growth</em> involves a number of ions, <em>growth</em> <em>factors</em> and cytokines and possibly additional but as yet unknown <em>factors</em>. Here we report on the potential role of the secretory N-terminal domain (sAPP) of the Alzheimer amyloid precursor protein (APP) in the regulation of <em>keratinocyte</em> proliferation. In human skin APP was detectable predominantly in the basal cell layer of the epidermis whereas the immunocytochemical signal in the underlying mesenchymal tissue was very low. Cultured normal human <em>keratinocytes</em> expressed the three APP isoforms 695, 751 and 770 with highest values for the isoforms 751 and 770. HaCaT cells, a spontaneously immortalized human <em>keratinocyte</em> cell line, exhibited almost identical patterns in the expression of the APP isoforms and in the release of endogenous sAPP. In HaCaT cells, recombinant sAPP (sAPPrec) was found to compete with endogenous sAPP for the same binding sites. Binding of sAPPrec was specific and occurred in microdomains of approximately 0.1 to approximately 0.3 microm in diameter. At 10 nM, sAPPrec binding induced a <em>2</em>- to 4-fold increase in the rate of cell <em>growth</em>. sAPP concentrations in the conditioned media were found to reach 5-<em>2</em>0 nM which is in the mitogenic range of sAPPrec. The proliferative effect of sAPP was inhibited by approximately 50% when antisense oligonucleotides directed against the APP mRNA were applied. The predominant expression of

<em>Growing</em> evidence has demonstrated the crucial role of NF-kappaB activation on disease severity in allergic disorders. In this study, we examined the clinical relevance of a novel NF-kappaB inhibitor, IMD-0354, for atopic dermatitis (AD) by its topical application. To investigate the in vivo efficacy, 1% IMD-0354 ointment was applied daily to NC/NgaTnd mice with severe dermatitis, which served as a model for human AD. During <em>2</em> weeks of treatment, scratching behavior decreased and severity of dermatitis reduced in mice treated with IMD-0354 as well as FK506 without diverse effects. Based on histological examinations, the hyperplasia of <em>keratinocytes</em> and infiltration of inflammatory cells were significantly reduced in the skin of IMD-0354-treated mice. The expressions of T-helper <em>2</em> cytokines and tumor necrosis <em>factor</em>-alpha at the affected skin sites were downregulated in IMD-0354-treated mice. Furthermore, IMD-0354 suppressed the proliferation of various immunocompetent cells, neurite out<em>growth</em> of nerve <em>growth</em> <em>factor</em>-stimulated pheochromocytoma cells, IgE production from splenic B cells, and IgE-mediated activation of mast cells in vitro. IMD-0354 effectively reduced the allergic inflammation in NC/NgaTnd mice in vivo. Thus, a drug that interferes with NF-kappaB activity may provide an alternative therapeutic strategy for the treatment of AD.