OBJECTIVE

To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells.

METHODS

Cell cultures of corneal <em>keratinocytes</em> (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.<em>2</em>5-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-<em>2</em>-yl)-<em>2</em>,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after <em>2</em>4 hours of incubation. Expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), VEGF receptors (VEGFR1 and VEGFR<em>2</em>), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after <em>2</em>4 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.<em>2</em>5-5.0 mg/mL), and signs of cellular damage were assessed.

RESULTS

No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR<em>2</em>. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS.

CONCLUSIONS

Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is <em>2</em>0-fold higher than that used for intravitreal application.

Bacillus anthracis lethal toxin (LT) is a key virulence <em>factor</em> of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn(<em>2</em>+)-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK<em>2</em> signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as <em>keratinocyte</em> <em>growth</em> <em>factor</em> or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.

Several proteases and their co-expressed inhibitors modulate the interdependent processes of cell migration and matrix proteolysis during wound repair. Transcription of the gene encoding plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor important in the control of barrier proteolysis and cell-to-matrix adhesion, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. Using a well-defined culture model of acute epidermal wounding and reepithelialization, PAI-1 mRNA/protein synthesis was induced early after monolayer scraping and restricted to cells comprising the motile cohort. PAI-1 levels in locomoting cells remained elevated (relative to the distal, contact-inhibited monolayer regions) throughout the time course of trauma repair. Targeted PAI-1 downregulation by transfection of antisense PAI-1 expression constructs significantly impaired <em>keratinocyte</em> migration and monolayer scrape wound closure. Injury-induced PAI-1 transcription closely paralleled <em>growth</em> state-dependent controls on the PAI-1 gene. An E-box motif (CACGTG) in the PAI-1 proximal promoter (located at nucleotides -160 to -165), previously shown to be necessary for serum-induced PAI-1 expression, was bound by nuclear <em>factors</em> from wound-stimulated but not quiescent, contact-inhibited, <em>keratinocytes</em>. UV crosslinking approaches to identify E-box-binding <em>factors</em> coupled with deoxyoligonucleotide affinity chromatography and gel retardation assays confirmed at least one major E-box-binding protein in both serum- and wound-activated cells to be USF-1, a member of the helix-loop-helix family of transcription <em>factors</em>. An intact hexanucleotide E-box motif was necessary and sufficient for USF-1 binding using nuclear extracts from both serum- and wound-simulated cells. Two species of immunoreactive USF-1 were identified by western blotting of total cellular lysates that corresponded to the previously characterized phosphorylated and non-phosphorylated forms of the protein. USF-1 isolated by PAI-1 promoter-DNA affinity chromatography was almost exclusively phosphorylated. Only a fraction of the total cellular USF-1 in proliferating cultures, by comparison, was phosphorylated at any given time. PAI-1 E-box binding activity, assessed by probe mobility shift criteria, increased within <em>2</em> hours of monolayer scrape injury, a time frame consistent with wound-stimulated increases in PAI-1 transcription. Relative to intact cultures, scrape site-juxtaposed cells had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity correlating with the specific in situ-restricted expression of PAI-1 transcripts/protein in the wound-edge cohort. USF-1 immunocytochemical staining declined significantly with increasing distance from the denudation site. These data are the first to indicate that binding of USF-1 to its target motif can be induced by 'tissue' injury in vitro and implicate USF-1 as a transcriptional regulator of genes (e.g. PAI-1) involved in wound repair.

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related <em>factor</em> protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary <em>keratinocytes</em> and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed <em>keratinocytes</em> and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized <em>keratinocytes</em> was approximately fourfold higher than in HPV-negative primary <em>keratinocytes</em>. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell <em>growth</em> within <em>2</em> weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed <em>2</em>5 and 40% <em>growth</em> inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.

Collagenase-3 (matrix metalloproteinase 13; MMP-13) is a novel matrix metalloproteinase, the expression of which to date has only been detected in human breast carcinoma tissue and osteoarthritic cartilage. Here, we show that MMP-13 transcripts are expressed by human HaCaT <em>keratinocytes</em> but not by primary human epidermal <em>keratinocytes</em>. The levels of MMP-13 mRNAs in HaCaT cells were enhanced up to 130- and 45-fold by tumor necrosis <em>factor</em> alpha (TNF-alpha) and transforming <em>growth</em> <em>factor</em> beta (TGF-beta), respectively. The maximal induction of MMP-13 mRNAs by TNF-alpha was noted after a 6-h incubation, whereas with TGF-beta, the maximal stimulation was observed after <em>2</em>4 h. The up-regulation of MMP-13 mRNA abundance by TNF-alpha and TGF-beta was dependent on protein synthesis and was prevented partially by dexamethasone and retinoic acid. Nuclear run-on assays demonstrated activation of MMP-13 gene transcription by TNF-alpha maximally at the <em>2</em>-h time point and by TGF-beta after 1<em>2</em> h of treatment. Incubation of HaCaT <em>keratinocytes</em> with TNF-alpha and TGF-beta also increased production of proMMP-13 into the culture media, as detected by Western blotting. Our data indicate that the MMP-13 gene is expressed by transformed epidermal <em>keratinocytes</em>, suggesting a role for MMP-13 in the invasive capacity of human epidermal malignancies.

The vasculature in adult skin remains normally quiescent, due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. However, skin retains the capacity for brisk initiation of angiogenesis, the <em>growth</em> of new blood vessels from preexisting vessels, during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and skin cancers such as cutaneous squamous cell carcinomas. Moreover, cyclic vascular expansion occurs during the <em>growth</em> phase of the hair follicle. Recent evidence suggests vascular endothelial <em>growth</em> <em>factor</em> as the major skin angiogenesis <em>factor</em>. During skin angiogenesis, expression of vascular endothelial <em>growth</em> <em>factor</em> is induced in epidermal <em>keratinocytes</em> by several stimuli including transforming <em>growth</em> <em>factor</em>-alpha and hypoxia, leading to increased vascularization of the dermis. In contrast, vascular endothelial <em>growth</em> <em>factor</em>-C induces skin lymphangiogenesis. Thrombospondin-1 and thrombospondin-<em>2</em> are endogenous inhibitors of angiogenesis that are expressed in normal skin, maintaining the quiescence of cutaneous vessels. Both inhibitors potently inhibit skin cancer <em>growth</em> via inhibition of tumor angiogenesis. Targeting cutaneous blood vessels represents a promising new therapeutic approach for the treatment of a variety of skin diseases.

Interactions between epidermal <em>keratinocytes</em> and dermal fibroblasts play an important role in regulating tissue homeostasis and repair. Nevertheless, little is known about the role of <em>keratinocytes</em> in the pathogenesis of keloid. In this study, we investigated the influence of normal skin- and keloid-derived <em>keratinocytes</em> on normal skin- and keloid-derived fibroblasts utilizing a serum-free indirect coculture system. The keloid-derived fibroblasts showed a greater proliferation and minimal apoptosis when cocultured with normal skin- or keloid-derived <em>keratinocytes</em>, and the results were most significant in the latter. This difference was not observed when the fibroblasts were treated with conditioned medium obtained from normal skin- and keloid-derived <em>keratinocytes</em>. Nevertheless, conditioned medium-treated groups showed more proliferation and less apoptosis compared to the nonconditioned medium-treated control groups. We also analyzed the profile of <em>factors</em> involved in cell <em>growth</em> and apoptosis in fibroblasts cocultured with <em>keratinocytes</em>. Extracellular signal-regulated kinase and c-Jun N-terminal kinase phosphorylations and expression of Bcl-<em>2</em> and transforming <em>growth</em> <em>factor</em>-beta1 were all significantly upregulated in the fibroblasts cocultured with keloid-derived <em>keratinocytes</em>. Together, these results strongly suggest that the overlying <em>keratinocytes</em> of the keloid lesion play an important role in keloidogenesis by promoting more proliferation and less apoptosis in the underlying fibroblasts through paracrine and double paracrine effects.

Hypoxia-inducible <em>factor</em>-1 (HIF-1) is a known cancer progression <em>factor</em>, promoting <em>growth</em>, spread, and metastasis. However, in selected contexts, HIF-1 is a tumor suppressor coordinating hypoxic cell cycle suppression and apoptosis. Prior studies focused on HIF-1 function in established malignancy; however, little is known about its role during the entire process of carcinogenesis from neoplasia induction to malignancy. Here, we tested HIF-1 gain of function during multistage murine skin chemical carcinogenesis in K14-HIF-1alpha(Pro40<em>2</em>A564G) (K14-HIF-1alphaDPM) transgenic mice. Transgenic papillomas appeared earlier and were more numerous (6 +/- 3 transgenic versus <em>2</em> +/- 1.5 nontransgenic papillomas per mouse), yet they were more differentiated, their proliferation was lower, and their malignant conversion was profoundly inhibited (7% in transgenic versus 40% in nontransgenic mice). Moreover, transgenic cancers maintained squamous differentiation whereas epithelial-mesenchymal transformation was frequent in nontransgenic malignancies. Transgenic basal <em>keratinocytes</em> up-regulated the HIF-1 target N-myc downstream regulated gene-1, a known tumor suppressor gene in human malignancy, and its expression was maintained in transgenic papillomas and cancer. We also discovered a novel HIF-1 target gene, selenium binding protein-1 (Selenbp1), a gene of unknown function whose expression is lost in human cancer. Thus, HIF-1 can function as a tumor suppressor through transactivation of genes that are themselves targets for negative selection in human cancers.

The fibroblast <em>growth</em> <em>factor</em> (FGF) family is a group of homologous heparin-binding polypeptides that has been implicated in a variety of human neoplasms and presently includes 14 members. FGF signaling is mediated by a dual-receptor system, consisting of four high-affinity tyrosine kinase receptors, termed fibroblast <em>growth</em> <em>factor</em> receptors (FGFRs), and of low-affinity heparan sulfate proteoglycan receptors that enhance ligand presentation to the FGFRs. Several FGFs, including FGF-1, -<em>2</em>, -3, -4, -5, -6, and -7, and several FGFR variants, among them the <em>2</em> immunoglobulin-like form and the IIIc splice variant of FGFR-1 and the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor, a splice variant of FGFR-<em>2</em>, are expressed in human pancreatic cancer cell lines and are overexpressed in human pancreatic cancers or in the pancreas of chronic pancreatitis and, therefore, may play important roles in the pathobiology of these pancreatic diseases. This review summarizes the current information on the involvement of the FGF family and their receptors in human pancreatic cancer and chronic pancreatitis.

BACKGROUND

Tumor-associated differentially expressed gene-15 (TADG-15/matriptase/MT-SP1) is a novel transmembrane serine protease involved in numerous biologic processes, including activation of growth and angiogenic factors and degradation of extracellular matrix components. To assess the value of TADG-15 as a possible marker for tumor detection and/or as a target for therapeutic intervention, the authors investigated the frequency of expression of TADG-15 in human cervical tumors.

METHODS

TADG-15 expression was evaluated in 19 cervical carcinoma cell lines (i.e., 11 primary tumor cell lines and 8 established cell lines) and in 8 normal cervical keratinocyte control cultures using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, to validate gene expression data at the protein level, TADG-15 expression was evaluated by immunohistochemistry on paraffin embedded tissue from which all 11 primary tumor cell lines were established.

RESULTS

TADG-15 was expressed at high levels in 8 of 11 (73%) primary cervical carcinoma cell lines and in 6 of 8 (75%) established cervical carcinoma cell lines by RT-PCR. Expression of TADG-15 was found in 6 of 6 (100%) primary squamous cell cervical carcinomas, whereas 2 of 5 (40%) primary adenocarcinomas expressed TADG-15. In contrast, none of the normal cervical keratinocyte control cultures (n = 4) or flash-frozen normal cervical biopsy specimens (n = 4) expressed TADG-15. Immunohistochemistry staining of paraffin embedded cervical carcinoma specimens confirmed TADG-15 expression in tumor cells and its absence on normal cervical epithelial cells.

CONCLUSIONS

Cervical carcinoma cells expressed high levels of TADG-15, suggesting that this protease may play an important role in invasion and metastasis. Because TADG-15 appears only in abundance in squamous tumor tissue and contains a proteolytic cleavage site, suggesting that the TADG-15 protease domain is released, it may prove to be a useful diagnostic tool for the early detection of recurrent/persistent cervical carcinoma after standard treatment or as a novel molecular target for therapy in patients with cervical carcinoma.

In this study we investigated the role of interleukin-15 (IL-15) in the immunobiology of cutaneous T-cell lymphoma (CTCL) cells. Using cell culture techniques, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunhistochemistry we found that IL-15, like IL-7, is a <em>growth</em> <em>factor</em> for the Sézary cell line SeAx and that both cytokines prolonged the survival of malignant T cells directly isolated from Sézary syndrome (SS) patients. Both IL-15 and IL-7 were more potent than IL-<em>2</em>. IL-4 and IL-9, whose receptors share the same gamma chain with the receptors of IL-<em>2</em>, IL-7, and IL-15, did not sustain the <em>growth</em> of CTCL cells, indicating that signaling through the common gamma chain (gammac) is not sufficient for continuous <em>growth</em>. IL-13 and tumor necrosis <em>factor</em>-alpha (TNF-alpha) had no effect. IL-7 and IL-15 also supported the <em>growth</em> of SeAx cells in the presence of the apoptosis inducing agents dexamethasone and retinoic acid. The analysis of patient Sézary cells and three CTCL cell lines by RT-PCR showed that all these cells expressed IL-15 mRNA, but only a few (<em>2</em>5%) produced IL-7 mRNA. Immunohistological analyses of skin biopsy samples of SS and Mycosis fungoides patients showed immunoreactivity for IL-15 in basal cell layer <em>keratinocytes</em> and in the infiltrating lymphocytes. We conclude that IL-15 is a <em>growth</em> or viability <em>factor</em> for CTCL-derived cell lines or shortly cultivated Sézary cells. The findings that IL-15 mRNA can be detected in Sézary syndrome peripheral blood mononuclear cells and that the IL-15 protein is detected in skin sections from CTCL patients suggest that IL-15 plays an important role in the biology of CTCL.

Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS<em>2</em> (hyaluronan synthase <em>2</em>). Increasing UDP-HexNAc in HaCaT <em>keratinocytes</em> by adding glucosamine down-regulated HAS<em>2</em> gene expression, whereas a decrease in UDP-HexNAc, realized by mannose treatment or siRNA for GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1), enhanced expression of the gene. Tracing the UDP-HexNAc-initiated signal to the HAS<em>2</em> promoter revealed no change in the binding of STAT3, NF-κB, and cAMP response element-binding protein, shown previously to mediate <em>growth</em> <em>factor</em> and cytokine signals on HAS<em>2</em> expression. Instead, altered binding of SP1 and YY1 to the promoter correlated with cellular UDP-HexNAc content and inhibition of HAS<em>2</em> expression. siRNA silencing of YY1 and SP1 confirmed their inhibitory effects on HAS<em>2</em> expression. Reduced and increased levels of O-GlcNAc-modified SP1 and YY1 proteins were associated with stimulation or inhibition of HAS<em>2</em> expression, respectively. Our data are consistent with the hypothesis that, by regulating the level of protein O-GlcNAc modifications, cellular UDP-HexNAc content controls HAS<em>2</em> transcription and decreases the effects on hyaluronan synthesis that would result from cellular fluctuations of this substrate.

Sensory neurons innervating the skin can release neuropeptides that are believed to modulate cellular proliferation, wound healing, pigmentation, and <em>keratinocyte</em> innate immune responses. While the ability of neuropeptides to stimulate <em>keratinocyte</em> production of inflammatory mediators has been demonstrated, there is no information concerning the mechanisms by which neuropeptide activation of <em>keratinocyte</em> cell surface receptors ultimately leads to the up-regulation of mediator production. In this study we used a <em>keratinocyte</em> cell line to identify the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) receptors on <em>keratinocytes</em> and examined the effects of SP and CGRP stimulation on <em>keratinocyte</em> neuropeptide signaling, cell proliferation, and interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis <em>factor</em> α (TNF-α), and nerve <em>growth</em> <em>factor</em> (NGF) expression. Neuropeptide stimulation caused an up-regulation of neuropeptide receptor expression in <em>keratinocytes</em> and a dramatic increase in <em>keratinocyte</em> secretion of SP and CGRP, suggesting possible autocrine or paracrine stimulatory effects and amplification of neuropeptide signaling. Both SP and CGRP concentration-dependently stimulated cellular proliferation and the expression and secretion of inflammatory cytokines and NGF in <em>keratinocytes</em>. SP also activated all 3 families of mitogen activated protein kinase (MAPK) and nuclear <em>factor</em> κB (NFκB) in <em>keratinocytes</em>, while CGRP only activated p38 and extracellular signal related kinase1/<em>2</em> (ERK1/<em>2</em>) MAPKs. Neuropeptide stimulated inflammatory mediatory production in <em>keratinocytes</em> was reversed by ERK1/<em>2</em> and JNK inhibitors. The current study is the first to observe; 1) that CGRP stimulates <em>keratinocyte</em> expression of CGRP and its receptor complex, <em>2</em>) that SP and CGRP stimulate IL-6 and TNF-α secretion in <em>keratinocytes</em>, 3) that SP activated all three MAPK families and the NFκB transcriptional signaling pathway in <em>keratinocytes</em>, and 4) that SP and CGRP stimulated inflammatory mediator production in <em>keratinocytes</em> is dependent on ERK1/<em>2</em> and JNK activation. These studies provide evidence suggesting that disruption of ERK1/<em>2</em> and JNK signaling may potentially be an effective therapy for inflammatory skin diseases and pain syndromes mediated by exaggerated sensory neuron-<em>keratinocyte</em> signaling.

Irradiation of human <em>keratinocytes</em> with UVB (<em>2</em>80-3<em>2</em>0 nm) in vitro and in vivo activates the metabolism of 7-dehydrocholesterol to hormonally active calcitriol. The production of calcitriol in the skin strongly depends on the photosynthesis of vitamin D(3) which is biologically inactive in the first instance. Vitamin D(3) serves as the starting substrate for two subsequent enzymatic hydroxylation steps in epidermal <em>keratinocytes</em>. Both the amount of vitamin D(3) and the activity of anabolic and catabolic vitamin D hydroxylases determine the cutaneous level of calcitriol. The hormonally active metabolite of vitamin D(3) regulates a huge number of genes in <em>keratinocytes</em>, and thus acts in an autocrine and/or paracrine manner. This local pathway of vitamin D(3) is unique, but its relevance for healthy and diseased skin is widely unknown, yet. Experimental findings implicate several questions: (1) Is UVB-induced formation of calcitriol involved in regulation of <em>growth</em> and differentaition of epidermal cells as well as immunological and skin protective processes? (<em>2</em>) What endogenous and exogenous <em>factors</em> including drugs affect the cutaneous vitamin D(3) pathway? From a therapeutical point of view, it has been known for a long time that topical application of calcitriol and its analogs can improve hyperproliferative skin diseases like psoriasis. In spite of many encouraging studies in recent years, the fields of the routinely therapeutical application of calcitriol or vitamin D analogs in dermatology (e.g. treatment of immunological, inflammatory, malignancies and infectious skin diseases) have not been intensified. Why is that?

We quantified the mRNA expression of all <em>2</em><em>2</em> fibroblast <em>growth</em> <em>factor</em> family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair <em>growth</em> cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, <em>2</em>, 5, 7, 10, 13, and <em>2</em><em>2</em>), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair <em>growth</em> cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and <em>2</em><em>2</em> at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal <em>keratinocytes</em> and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair <em>growth</em> was observed. Our findings suggest that FGF18 is important for the regulation of hair <em>growth</em> and the maintenance of skin in adult mice.

gamma delta T cells are a distinct subgroup of T lymphocytes that are enriched at certain anatomical localizations, such as the small intestinal epithelia and other epithelia. gamma delta T cells recognize microbial antigens, such as heat shock proteins (in mice) or phosphorylated bacterial metabolites (in humans), and control the integrity of epithelia. At the effector cell level, they share with the conventional alpha beta T lymphocytes potent cytotoxic activity and the capacity to produce a variety of cytokines, including specific cytokines such as <em>keratinocyte</em> <em>growth</em> <em>factor</em>. Here we summarize the current knowledge on the role of chemokines and their receptors in the migration and function of gamma delta T cells. As an example, the migration of gamma delta T cells to the small intestine is guided by the chemokine receptor CCR9 and the local expression of the corresponding ligand CCL<em>2</em>5 (also termed thymus-expressed chemokine, TECK). Chemokine receptor expression also correlates with the functional program of T cells. In this respect, the strong expression of the MIP-1 alpha/MIP-1 beta/RANTES (CCL3/CCL4/CCL5)-receptor CCR5 correlates with a T-helper 1 phenotype of human V gamma 9V delta <em>2</em>-expressing gamma delta T cells. The regulation of chemokine receptors, together with the pattern of local chemokine production, plays an important role in the localization of gamma delta T cells under physiological and pathophysiological conditions, such as infection, inflammation, and tumor defense.

Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 13<em>2</em> genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique <em>factors</em> that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial <em>growth</em> <em>factor</em> genes have been "captured" from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating <em>factor</em> and interleukin-<em>2</em> appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear <em>factor</em> (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in <em>keratinocytes</em>. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis.

UVB irradiation of epidermal <em>keratinocytes</em> results in the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and subsequently activator protein-1 (AP-1) transcription <em>factor</em> activation and cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression. AP-1 and COX-<em>2</em> have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-<em>2</em> expression, and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-<em>2</em> expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and <em>growth</em> compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and <em>growth</em> rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of <em>keratinocytes</em>. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV-induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-<em>2</em> expression and proliferation of UVB-irradiated cells.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a potent mitogen of alveolar epithelial type II cells (AEII). AEII hyperplasia is resolved within several days following intratracheal instillation of KGF by unknown mechanism(s). AEII hyperplasia was induced in rat lungs by intrabronchial instillation of 5 mg recombinant human (rh)KGF x kg body weight(-1) or an equivalent amount of diluent. Epithelial architecture, cell proliferation, transformation of AEII into type I cells (AEI) and apoptosis were investigated by means of immunohistochemistry, stereology, double immunofluorescence microscopy, electron microscopy and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) technique in lungs fixed 1, <em>2</em>, 3 and 7 days after treatment. After 1 day of rhKGF instillation, an increase was observed in the nuclear antigen Ki-67, a proliferation marker detected by the antibody MIB-5-expressing surfactant protein (SP)-B, -C, -D-positive AEII. The incidence of mitosis was increased by day <em>2</em>, resulting in AEII micropapillae with intense basolateral expression of the exon 6 containing isoform (v6) of CD446 (CD44v6), a marker for AEII. By day 3, monolayers of AEII exhibiting lateral CD44v6 covered 45% of the alveolar surface. After 7 days, there were numerous intermediate AEII/AEI cells characterized by a flat elongated shape, staining for SP-D, apical appearance of AEI marker Lycopersicon esculentum lectin and lateral staining for AEII marker CD44v6. Increased numbers of TUNEL-positive epithelial cells were seen at days <em>2</em>-7. In conclusion, restoration of normal alveolar epithelium after instillation of recombinant human <em>keratinocyte</em> <em>growth</em> <em>factor</em> is accomplished by terminal differentiation and apoptosis of hyperplastic alveolar epithelial type II cells in vivo.

Intracellular signaling induced by the coagulation <em>factors</em> (F) VIIa and Xa is poorly understood. We report here studies on these processes in a human <em>keratinocyte</em> line (HaCaT), which is a constitutive producer of tissue <em>factor</em> (TF) and responds to both FVIIa and FXa with elevation of cytosolic Ca(<em>2</em>+), phosphorylation of extracellular signal-regulated kinase (Erk) 1/<em>2</em>, p38(MAPK), and c-Jun N-terminal kinase, and up-regulation of transcription of the early <em>growth</em> response gene-1 (egr-1). Using egr-1 as end point, we observed with both agonists that phosphatidylinositol-specific phospholipase C and the mitogen-activated protein kinase/Erk kinase/Erk pathway were mediators of the responses. The responses to FVIIa were TF-dependent and up-regulation of egr-1 mRNA did not require presence of the TF cytoplasmic domain. Antibodies to EPR-1 and <em>factor</em> V had no effect on the response to FXa. We have provided evidence that TF is not the sole component of the FVIIa receptor. The requirement for proteolytic activity of both FVIIa and FXa suggests that protease-activated receptors may be involved. We now report evidence suggesting that protease-activated receptor <em>2</em> or a close homologue may be a necessary but not sufficient component of this particular signal transduction pathway. The up-regulation of egr-1 describes one way by which the initiation of blood coagulation may influence gene transcription. The ability of these coagulation proteases to induce intracellular signals at concentrations at or below the plasma concentrations of their zymogen precursors suggests that these processes may occur also in vivo.

<em>Keratinocyte</em> differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl(-) intracellular channel 4 (CLIC4) expression is increased in both mouse and human <em>keratinocytes</em> undergoing differentiation induced by Ca(<em>2</em>+), serum and the protein kinase C (PKC)-activator, 1<em>2</em>-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCdelta, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca(<em>2</em>+)-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating <em>keratinocytes</em>. CLIC4 is cytoplasmic in actively proliferating <em>keratinocytes</em> in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in <em>keratinocytes</em> undergoing <em>growth</em> arrest by differentiation, senescence or transforming <em>growth</em> <em>factor</em> beta (TGFbeta) treatment. Targeting CLIC4 to the nucleus of <em>keratinocytes</em> via adenoviral transduction increases nuclear Cl(-) content and enhances expression of differentiation markers in the absence of elevated Ca(<em>2</em>+). In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl(-) and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with <em>keratinocyte</em> terminal differentiation.

p63 is a developmentally regulated transcription <em>factor</em> related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon-Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G(<em>2</em>)/M genes by binding to multiple CCAAT core promoters in immortalized and primary <em>keratinocytes</em>. The CCAAT-activator NF-Y and DeltaNp63alpha are associated in vivo and a conserved alpha-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. DeltaNp63alpha, but not the AEC mutants repress CCAAT-dependent transcription of G(<em>2</em>)/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G(<em>2</em>)/M promoters, while normally present on 14-3-3sigma, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon <em>keratinocytes</em> differentiation, NF-Y and p63 remain bound to G(<em>2</em>)/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of <em>growth</em> activating genes upon differentiation is compromised by AEC missense mutations.

Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human <em>keratinocytes</em> in part by inducing oxidative stress and cytokine production. Severe UVB damage to the <em>keratinocyte</em> can also result in apoptosis or programmed cell death. Although the lipid mediator platelet-activating <em>factor</em> (PAF) is synthesized in response to epidermal cell damage and epidermal cells express PAF receptors, it is not known whether PAF is involved in UVB-induced epidermal cell apoptosis. These studies examined the role of the PAF system in UVB-induced epidermal cell apoptosis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Expression of the PAF-R in KB cells did not affect base-line <em>growth</em> or apoptosis, yet resulted in a decrease in the lag time between treatment of the cells and the induction of apoptosis following irradiation with 400 J/m<em>2</em> UVB. This effect was inhibited by pretreatment with the PAF-R antagonists WEB <em>2</em>086 and A-85783, confirming involvement of the PAF-R in this process. At lower doses (100-<em>2</em>00 J/m<em>2</em>) of UVB, only KB cells that expressed the PAF-R became apoptotic. Treatment of PAF-R-expressing KB clones with the metabolically stable PAF-R agonist 1-hexadexyl-<em>2</em>-N-methylcarbamoyl-3-glycerophosphocholine (CPAF) alone did not induce apoptosis but augmented the degree of apoptosis observed if CPAF was used in combination with lower doses (<em>2</em>00 J/m<em>2</em>) of UVB irradiation. Interestingly, UVB irradiation was found to stimulate PAF synthesis only in PAF-R-expressing KB cell clones. The antioxidants N-acetyl cysteine, 1,1,3,3-tetramethyl-<em>2</em>-thiourea, and vitamin E inhibited both UVB-induced PAF biosynthesis as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB clones, suggesting the possibility that UVB stimulates the production of oxidized lipid species with PAF-R agonistic activity in this model system. Thus, these studies indicate that a component of UVB-induced epidermal cell cytotoxicity can be modulated by PAF-R activation through the production of PAF and PAF-like species.

About 600,000 people in the United States are estimated to be affected by venous ulcers. The cornerstone of care of chronic venous ulcers involves the application of compression bandages. Other therapies include treatment of associated infection, treatment for edema and inflammation, and debridement when necessary. Repifermin, a recombinant human KGF-<em>2</em> (fibroblast <em>growth</em> <em>factor</em>-10), exerts a proliferative effect on epithelial cells, in vitro and in vivo, and has been shown to accelerate wound healing in several experimental animal models. A randomized, double-blind, parallel-group, placebo-controlled, multicenter study was conducted to evaluate the safety and efficacy of topical repifermin treatment, for 1<em>2</em> weeks, in the healing of chronic venous ulcers in 94 patients. Repifermin was shown to accelerate wound healing, with significantly more patients achieving 75% wound closure with repifermin than with placebo. The treatment effect appeared more marked for a subgroup of patients with initial wound areas < or = 15 cm<em>2</em> and wound ages of < or = 18 months. A longer duration of treatment (e.g., <em>2</em>6 weeks) may allow better differentiation of the benefit of repifermin compared with placebo, particularly with respect to complete wound closure. The safety assessment showed that repifermin was well tolerated.