Wound healing is a complex process that relies on proper levels of cytokines and <em>growth</em> <em>factors</em> to successfully repair the tissue. Of particular interest are the members of the transforming <em>growth</em> <em>factor</em> family. There are three TGF-ß isoforms-TGF- ß 1, <em>2</em>, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and <em>keratinocyte</em> migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in <em>keratinocyte</em> migration. Wild type <em>keratinocytes</em> treated with NAB and Tgfb3-deficient <em>keratinocytes</em> closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.

The investigation of novel targets for the treatment of cystic fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistent with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in mice and patients with CF, SLs were identified as targets for treating pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the Pseudomonas aeruginosa-dependent transcription of the IL-8 gene in bronchial cells, we examined the effects of miglustat and amitriptyline, another drug affecting ceramide metabolism, on the expression of 9<em>2</em> genes implicated in host immune defense. Infection with the P. aeruginosa strain PAO1 up-modulated the expression of 14 (<em>2</em>7%) genes in IB3-1 cells and 15 (<em>2</em>9%) genes in CF primary respiratory epithelia grown at an air-liquid interface, including chemokines (IL-8, <em>growth</em>-regulated Gro-α/β/γ proteins, and granulocyte chemotactic peptide-<em>2</em> [GCP-<em>2</em>]), proinflammatory cytokines (IL-1α/β, IL-6, and TNF-α), and the intercellular adhesion molecule-1, nuclear <em>factor</em> kB1, toll like receptor <em>2</em>, and human defensin B4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the <em>keratinocyte</em>-derived chemokine in lung extracts. Collectively, these results indicate that targeting the metabolism of SLs can down-modulate the recruitment of neutrophils into the lung.

BACKGROUND

Rosacea is a common chronic disease of unclear pathogenesis, characterised by inflammation and vascular abnormalities of the facial skin and ocular surface. Recognising that vascular endothelial growth factor (VEGF) is vasoactive and has inflammatory activities, the expression of this molecule and its receptors, VEGF-R1 and VEGF-R2, in rosacea was investigated.

METHODS

Formalin-fixed, paraffin wax-embedded sections of skin obtained from 20 patients with rosacea were immunostained to detect expression of VEGF, VEGF-R1 and VEGF-R2, using an indirect methodology incorporating antigen retrieval. Adjacent sections were stained with haematoxylin and eosin.

RESULTS

Biopsy specimens were characterised by perivascular and perifollicular lymphohistiocytic infiltration and dilated vascular channels. In addition to keratinocyte and epithelial staining, which was also noted in normal skin, vascular endothelium frequently stained positive for VEGF-R1 (14/20, 70%) and VEGF-R2 (20/20, 100%), but infrequently for VEGF (2/20, 10%). In most specimens, infiltrating leucocytes, including lymphocytes, macrophages and plasma cells, expressed VEGF (17/20, 85%), VEGF-R1 (20/20, 100%) and VEGF-R2 (20/20, 100%).

CONCLUSIONS

Expression of VEGF receptors, both by vascular endothelium and infiltrating mononuclear cells, is observed in rosacea. Although not expressed by endothelium, VEGF is present in epidermis and epithelium, and is expressed by infiltrating cells. VEGF receptor-ligand binding may contribute to the vascular changes and cellular infiltration that occurs in rosacea.

Tumor necrosis <em>factor</em>-alpha (TNF-alpha) plays a central role in sustaining the inflammatory process in the skin as well as in the joints of patients with psoriasis and psoriatic arthritis. In fact, biological therapies based on monoclonal antibodies against TNF-alpha have been proven to be effective on both the arthropathy and the cutaneous symptoms of the disease. Among the several effects produced by TNF-alpha on <em>keratinocytes</em> there is the induction of expression of MMP-9, a matrix metalloproteinase (MMP) produced mainly by monocytes and macrophages. In this article we refer to the results of a study on the behavior of MMP-9 in the sera and in the lesional skin in association with effective therapy with infliximab. Measurements of TNF-alpha, MMP-<em>2</em>, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and E-selectin were also performed in the same samples. Eleven psoriatic patients included in a therapeutic protocol based on the administration of infliximab monotherapy were collected before treatment and after 6 and 1<em>2</em> weeks of therapy. Significant decrease of MMP-9 and MMP-<em>2</em> levels in the sera was associated with clinical improvement and with the decrease of TNF-alpha, VEGF, and E-selectin, angiogenic molecules already known to be implicated in the clinical expression of psoriasis. The clinical amelioration of the cutaneous expression of psoriasis was significantly associated with the decrease of MMP-9, TNF-alpha, and E-selectin levels, spontaneously released by lesional biopsy samples before and after therapy, measured in the culture supernatants by immunoenzymatic assays. In addition, significant correlations were found between the clinical score and TNF-alpha, MMP-9, and E-selectin lesional production. MMP-9 levels were significantly correlated with those of TNF-alpha. Our findings show the existence of a direct relationship between MMP-9 and TNF-alpha production, strongly suggesting that MMP-9 may play a key role in the skin inflammatory process in psoriasis, while a different role may be attributed to MMP-<em>2</em>.

We have previously shown that human squamous cell carcinomas (SCC) express the interleukin <em>2</em> receptor (IL<em>2</em>R)-alpha and -beta chains, and that the ligand, IL<em>2</em>, directly inhibits <em>growth</em> of the tumor in vitro and in vivo in the tumor xenograft-nude mice model. We now show that the alpha and beta chains of IL<em>2</em>R are expressed on a variety of human carcinoma cell lines and on normal human <em>keratinocytes</em> in early-stage cultures. While all carcinoma cells in a population expressed IL<em>2</em>R-alpha and -beta proteins, in <em>keratinocytes</em> obtained from different normal donors, variable proportions of cells were positive, as measured by flow cytometry. The carcinoma lines and <em>2</em>/5 <em>keratinocyte</em> lines studied were also found to contain transcripts for the IL<em>2</em>R-gamma chain detectable by combined reverse transcription-PCR (RT-PCR) and hybridization with the specific cDNA probe. Incubation of the gastric (HR) or renal cell carcinoma (RCC) cell lines, but not of other IL<em>2</em>R+ carcinoma cell lines or normal <em>keratinocytes</em>, in the presence of IL<em>2</em> resulted in dose-dependent inhibition of tumor cell <em>growth</em>. Monoclonal antibodies (MAbs) specific for IL<em>2</em>R-beta chain completely reversed this <em>growth</em> inhibitory effect of IL<em>2</em>. The ligand, IL<em>2</em>, also down-regulated surface expression of its own receptor and of intercellular adhesion molecule-I (ICAM-I) or class I major histocompatibility complex (MHC) antigens on IL<em>2</em>R+ tumor cells. All carcinoma cells studied incubated in the presence of IL<em>2</em> exhibited significantly increased sensitivity to <em>growth</em>-inhibitory effects of other cytokines such as interferon (IFN)-gamma, tumor necrosis <em>factor</em> (TNF)-alpha or transforming <em>growth</em> <em>factor</em> (TGF)-beta. IL<em>2</em> inhibited <em>growth</em> of the HR cells by arresting a significant proportion of tumor cells in the G0/G1 phase of the cell cycle. Thus, IL<em>2</em> can have direct effects on IL<em>2</em>R+ carcinoma cells, leading to changes in <em>growth</em> or to increases in sensitivity of tumor cells to cytostatic activities of other cytokines.

Quantification of <em>2</em>7 cytokines following cerebral wounding was performed for wound age estimation. The cytokines evaluated included interleukin (IL)-1alpha, IL-1beta, IL-<em>2</em>, IL-3, IL-4, IL-5, IL-6, IL-10, IL-1<em>2</em> p40, IL-1<em>2</em> p70, IL-15, IL-17, IL-18, basic fibroblast <em>growth</em> <em>factor</em> (bFGF), granulocyte-colony stimulating <em>factor</em> (G-CSF), granulocyte macrophage-colony stimulating <em>factor</em> (GM-CSF), Interferon-gamma (IFN-gamma), <em>keratinocyte</em> derived cytokine (KC), leukemia inhibitory <em>factor</em> (LIF), macrophage-colony stimulating <em>factor</em> (M-CSF), monokine inducible by interferon gamma (MIG), macrophage inflammatory protein (MIP)-1 alpha, MIP <em>2</em>, platelet-derived <em>growth</em> <em>factor</em> BB (PDGF BB), regulated upon activation, normal T-cell expressed, and secreted (Rantes), tumor necrosis <em>factor</em>-alpha (TNF-alpha), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The proliferation of glial cells as well as the infiltration of inflammatory cells were also evaluated. Although astroglia proliferated from 7<em>2</em> hours post-injury, inflammatory cell dynamics were generally steady. Among cytokines analyzed in the present study, IL-1beta, IL-5, IL-6, IL-1<em>2</em> p40, G-CSF, IFN-gamma, KC, LIF, MIP<em>2</em>, and PDGF BB increased during the early phase of cerebral wound healing, and M-CSF increased during the middle phase, while IL-15, IL-18, and MIG increased during the late phase. In contrast, IL-1alpha, IL-10, IL-1<em>2</em> p70, and TNF-alpha were suppressed throughout the cerebral wound healing process. Based on our findings, quantitative cytokine analyses at the cerebral wound site may be a useful tool for wound age estimation. Further, this study suggests that multiplex data gained from the same sample using a single methodology demonstrates highly accurate cytokine interactions during the process of cerebral wound healing.

Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) prevents lung damage and mortality in animals exposed to various forms of oxidant stress, but the protective mechanisms are not yet established. Because DNA strand break (DNA-SB) formation is one of the earliest cellular changes that occurs after cells are exposed to an oxidant stress, we determined whether KGF reduces H<em>2</em>O<em>2</em>-induced pulmonary toxicity by attenuating AEC DNA damage. KGF (10-100 ng/ml) decreased H<em>2</em>O<em>2</em> (0.05-0.5 mM)-induced DNA-SB formation in cultured A549 and rat alveolar type II cells measured by an alkaline unwinding, ethidium bromide fluorometric technique. The protective effects of KGF were independent of alterations in catalase, glutathione (GSH), or the expression of bcl-<em>2</em> and bax, two protooncogenes known to regulate oxidant-induced apoptosis. Actinomycin D and cycloheximide abrogated protective effects of KGF. Furthermore, protection by KGF was completely blocked by 1) genistein, a tyrosine kinase inhibitor; <em>2</em>) staurosporine and calphostin C, protein kinase C (PKC) inhibitors; and 3) aphidicolin, butylphenyl dGTP, and <em>2</em>',3'-dideoxythymidine 5'-triphosphate, inhibitors of DNA polymerase. We conclude that KGF attenuates H<em>2</em>O<em>2</em>-induced DNA-SB formation in cultured AECs by mechanisms that involve tyrosine kinase, PKC, and DNA polymerases. These data suggest that the ability of KGF to protect against oxidant-induced lung injury is partly due to enhanced AEC DNA repair.

Rat alveolar epithelial cells (AEC) in primary culture transdifferentiate from a type II (AT<em>2</em>) toward a type I (AT1) cell-like phenotype, a process that can be both prevented and reversed by <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). Microarray analysis revealed that these effects of KGF are associated with up-regulation of key molecules in the mitogen-activated protein kinase (MAPK) pathway. To further explore the role of three key MAPK (i.e., extracellular signal-related kinase [ERK] 1/<em>2</em>, c-Jun N-terminal kinase [JNK] and p38) in mediating effects of KGF on AEC phenotype, primary rat AEC cultivated in minimal defined serum-free medium (MDSF) were treated with KGF (10 ng/ml) from Day 4 for intervals up to 48 hours. Exposure to KGF activated all three MAPK, JNK, ERK1/<em>2</em>, and p38. Inhibition of JNK, but not of ERK1/<em>2</em> or p38, abrogated the ability of KGF to maintain the AT<em>2</em> cell phenotype, as evidenced by loss of expression of lamellar membrane protein (p180) and increased reactivity with the AT1 cell-specific monoclonal antibody VIIIB<em>2</em> by Day 6 in culture. Overexpression of JNKK<em>2</em>, upstream kinase of JNK, increased activation of endogenous c-Jun in association with increased expression of p180 and abrogation of AQP5, suggesting that activation of c-Jun promotes retention of the AT<em>2</em> cell phenotype. These results indicate that retention of the AT<em>2</em> cell phenotype by KGF involves c-Jun and suggest that activation of c-Jun kinase may be an important determinant of maintenance of AT<em>2</em> cell phenotype.

Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). We expressed PKCalpha fused to green fluorescent protein and examined its real time translocation to the plasma membrane in living human corneal epithelial cells. Upon 10 min of stimulation with epidermal and hepatocyte <em>growth</em> <em>factors</em> (EGF and HGF), PKCalpha translocated to the plasma membrane. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> did not stimulate PKCalpha translocation up to 1 h after stimulation. Pretreatment with the 15-lipoxygenase metabolite, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), followed by EGF or HGF, produced faster translocation of PKCalpha detectable at <em>2</em> min. However, the same concentration of 15(S)-HETE alone did not stimulate translocation. 15(S)-Hydroperoxyeicosatetraenoic acid and 5(S)-HETE did not affect <em>growth</em> <em>factor</em>-induced translocation of PKCalpha. PD153035, a specific inhibitor of tyrosine kinase activity of the EGF receptor, completely blocked PKCalpha translocation induced by EGF. PD98059, a specific MEK inhibitor, significantly inhibited EGF- and HGF-mediated PKCalpha translocation, which was reversed by addition of 15(S)-HETE. Phosphorylation of ERK1/<em>2</em> by EGF was followed by phosphorylation of cytosolic phospholipase A(<em>2</em>) (cPLA(<em>2</em>)), and blocking ERK1/<em>2</em> inhibited cPLA(<em>2</em>) activation. Immunofluorescence demonstrated translocation of p-cPLA(<em>2</em>) to plasma and nuclear membranes as early as <em>2</em> min. This may further increase arachidonic acid release from membrane phospholipid pools and increase the intracellular pool of HETEs. In fact, in cells prelabeled with [(3)H]arachidonic acid, EGF stimulated synthesis of 15(S)-HETE in the cytosolic fraction. 15(S)-HETE also reversed the effect of LOX inhibitor on EGF-mediated cell proliferation. Our results indicate that 15(S)-HETE is an intracellular second messenger that facilitates translocation of PKCalpha to the membrane and elucidate a mechanism that plays a regulatory role in cell proliferation crucial to corneal wound healing.

The effect of transforming <em>growth</em> <em>factor</em>-type beta 1(TGF-beta) on the <em>growth</em> and differentiation of normal human skin <em>keratinocytes</em> cultured in serum-free medium was investigated. TGF-beta markedly inhibited the <em>growth</em> of <em>keratinocytes</em> at the concentrations greater than <em>2</em> ng/ml under low Ca<em>2</em>+ conditions (0.1 mM). <em>Growth</em> inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main <em>factors</em> in the process of TGF-beta-induced <em>growth</em> inhibition of human <em>keratinocytes</em>. Since <em>growth</em> inhibition and induction of differentiation are closely related in human <em>keratinocytes</em>, the <em>growth</em>-inhibitory effect of TGF-beta under high Ca<em>2</em>+ conditions (1.8 mM Ca<em>2</em>+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the <em>growth</em> of <em>keratinocytes</em> under high Ca<em>2</em>+ conditions in the same manner as under low Ca<em>2</em>+ conditions, suggesting that it is a strong <em>growth</em> inhibitor in both low and high Ca<em>2</em>+ environments. The induction of <em>keratinocyte</em> differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at <em>2</em>0 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca<em>2</em>+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca<em>2</em>+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at <em>2</em>0 ng/ml decreased cornified envelope formation by 53% under low Ca<em>2</em>+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca<em>2</em>+ conditions. Thus, the effect of TGF-beta on <em>keratinocyte</em> differentiation is Ca<em>2</em>+ dependent. It enhances differentiation of human <em>keratinocytes</em> under high Ca<em>2</em>+ conditions, but inhibits differentiation under low Ca<em>2</em>+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on <em>growth</em> inhibition and induction of differentiation in human <em>keratinocytes</em>. These data indicate that <em>growth</em> inhibition of human <em>keratinocytes</em> by TGF-beta is direct and not induced by differentiation.

Neuroimmune dysregulation characterizes atopic disease, but its nature and clinical impact remain ill-defined. Induced by stress, the neurotrophin nerve <em>growth</em> <em>factor</em> (NGF) may worsen cutaneous inflammation. We therefore studied the role of NGF in the cutaneous stress response in a mouse model for atopic dermatitis-like allergic dermatitis (AlD). Combining several methods, we found that stress increased cutaneous but not serum or hypothalamic NGF in telogen mice. Microarray analysis showed increased mRNAs of inflammatory and <em>growth</em> <em>factors</em> associated with NGF in the skin. In stress-worsened AlD, NGF-neutralizing antibodies markedly reduced epidermal thickening together with NGF, neurotrophin receptor (tyrosine kinase A and p75 neurotrophin receptor), and transforming <em>growth</em> <em>factor</em>-β expression by <em>keratinocytes</em> but did not alter transepidermal water loss. Moreover, NGF expression by mast cells was reduced; this corresponded to reduced cutaneous tumor necrosis <em>factor</em>-α (TNF-α) mRNA levels but not to changes in mast cell degranulation or in the T helper type 1 (Th1)/Th<em>2</em> cytokine balance. Also, eosinophils expressed TNF receptor type <em>2</em>, and we observed reduced eosinophil infiltration after treatment with NGF-neutralizing antibodies. We thus conclude that NGF acts as a local stress mediator in perceived stress and allergy and that increased NGF message contributes to worsening of cutaneous inflammation mainly by enhancing epidermal hyperplasia, pro-allergic cytokine induction, and allergy-characteristic cellular infiltration.

Apigenin, a flavonoid abundant in various vegetables and fruits, including parsley and onions, has been reported to possess anticarcinogenic effects. However, the direct molecular target of apigenin and its chemopreventive effect on ultraviolet (UV)-induced skin inflammation are not understood fully. Herein, we examined the anti-inflammatory effect of apigenin and its associated mechanisms in JB6 P+ cell line and SKH-1 hairless mouse model. Apigenin inhibited UVB-induced cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression, which is a well-known key mediator of inflammation and cancer, and restored the upstream stimulatory <em>factor</em> level in JB6 P+ cells. Immunoblot and kinase assay data demonstrate that Src activity was attenuated by apigenin, and this led to subsequent inhibition of UVB-induced phosphorylation of epidermal <em>growth</em> <em>factor</em> receptor, mitogen-activated protein kinases and Akt signaling. Inhibitory effects of apigenin on UVB-induced signaling were also confirmed in HaCaT human <em>keratinocytes</em>. In addition, in vitro pull-down assays revealed that apigenin binds Src in an adenosine triphosphate-competitive manner. Results using in vivo skin model indicate apigenin significantly inhibits UVB-induced ear edema development, COX-<em>2</em> expression and Src kinase activity in SKH-1 hairless mice. Collectively, these findings suggest that apigenin exerts potent chemopreventive activity against UVB-induced skin inflammation primarily by targeting Src.

BACKGROUND

Human full-length keratinocyte growth factor (KGF) promotes healing of colon anastomoses in rats through mechanisms other than enhancement of collagen synthesis. Since insulin-like growth factor (IGF) I increases matrix synthesis, the aim of this study was to evaluate the effect of systemic truncated KGF (tKGF), IGF-I and combined tKGF-IGF-I administration on the healing of colonic anastomoses in rats.

METHODS

Rats underwent laparotomy, division of the left colon, and sigmoidosigmoidostomy. tKGF (1 mg/kg), IGF-I (1 mg/kg), tKGF-IGF-I (both 1 mg/kg) or vehicle was administered intraperitoneally in four groups (n = 18 per group) 12 h before surgical intervention, and then once daily until killing (six animals per group; 2, 4 and 6 days after surgery). Bursting pressure measurements, histological evaluation, morphometric analysis, mucin and collagen staining, and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry of the anastomotic site were undertaken.

RESULTS

Administration of tKGF, IGF-I and the combination of both growth factors significantly increased anastomotic bursting pressure at postoperative day 2 (63, 71 and 113 per cent respectively), day 4 (68, 83 and 80 per cent) and day 6 (48, 43 and 43 per cent) compared with the control group. No intergroup differences were found. Histological examination, mucin and BrdU staining, and measurement of colonic crypt depth indicated less inflammation, increased acidic mucin content, a higher crypt cell proliferation rate and thickened mucosal layer in the growth factor-treated animals than in controls. Enhanced collagen staining was observed only in IGF-treated animals.

CONCLUSIONS

tKGF and IGF-I markedly accelerate the healing of colonic anastomoses in rats. However, combined administration of the two growth factors does not show additional benefit. Both growth factors may be acting to accelerate host reparative processes as well as to enhance protection of the anastomotic wound bed.

Trefoil peptides are a family of small proteins expressed by goblet cells that are secreted onto the apical gastrointestinal mucosal surface, where they are present in high concentrations. These peptides appear to both protect the epithelium and promote healing after injury. However, the <em>factors</em> regulating the expression and secretion of these proteins contributing to mucosal defense have not been characterized. To determine the mechanisms controlling production of trefoil peptides, the human colon cancer-derived model cell line HT-<em>2</em>9 was exposed to a variety of potential secretagogues. Expression and secretion of human intestinal trefoil <em>factor</em> (hITF) as well as the intestinal apomucin MUC<em>2</em> were assessed by Northern and Western blot analysis. Carbachol, an analog of acetylcholine, and the neuroendocrine peptides somatostatin and vasoactive intestinal polypeptide (VIP) stimulated increased expression of hITF mRNA within 5 min. These same <em>factors</em> stimulated parallel secretion of the hITF peptide, with maximal stimulation observed at concentrations ranging from 10(-6) M (carbachol and somatostatin) to 10(-7) M (VIP). Expression and secretion of hITF in response to carbachol, VIP, and somatostatin was independent of production of apomucin. hITF was not regulated by other neuroendocrine transmitters including histamine and substance P. Similarly, hITF expression and secretion was not modulated by peptide <em>growth</em> <em>factors</em> (epidermal <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta, and <em>keratinocyte</em> <em>growth</em> <em>factor</em>), cytokines [interleukin (IL)-1 beta, IL-<em>2</em>, IL-7, and IL-11], or arachidonic acid metabolites (prostaglandin E1/E<em>2</em> and leukotriene B4). In conclusion, trefoil peptides appear to be integrated into mechanisms of mucosal defense and repair through the enteric neuroendocrine system and independent of the classical mucosal immune cytokine network.

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast <em>growth</em> <em>factor</em>-<em>2</em> (FGF-<em>2</em>), FGF-7 and epidermal <em>growth</em> <em>factor</em> (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. <em>Growth</em> <em>factors</em> produced by both methods were functional, inducing the <em>growth</em> of fibroblast cells and <em>keratinocytes</em>. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.

BACKGROUND

Ultraviolet (UV) B light is an environmental mutagen that induces changes in cutaneous gene expression leading to immune suppression and carcinogenesis. Keratinocytes are a primary target for UVB.

OBJECTIVE

To further delineate UVB-induced gene expression changes in keratinocytes.

METHODS

cDNA microarray technology was utilized to examine gene expression in normal human KC (NHKC) following 20 mJcm(-2) UVB irradiation. Data was confirmed by semi-quantitative RT-PCR.

RESULTS

Microarray analysis revealed 57 genes were upregulated, and 27 genes were downregulated, by at least two-fold following UVB. One downregulated gene was the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1). Semi-quantitative RT-PCR confirmed persistent downregulation of TSP-1 up to 18h following UVB. Microarray analysis also revealed upregulation of platelet-derived endothelial cell growth factor (PD-ECGF)--an angiogenesis activator.

CONCLUSIONS

Our results suggest a gene expression mechanism by which UVB induces an angiogenic switch in keratinocytes. This may represent an important early event promoting neovascularization and growth of cutaneous neoplasms.

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

Inorganic arsenic is a ubiquitous environmental contaminant associated with an increased risk of skin hyperkeratosis and cancer. Although several hypotheses that relate to arsenic-induced carcinogenesis have been suggested, the mechanism of action remains obscure. In the present study, molecular mechanisms underlying the inactivation of p53 function and the genomic instability in malignant transformation of the human <em>keratinocyte</em> cell line, HaCaT, induced by low levels of arsenic were investigated. Our results show that long-term exposure of HaCaT cells to sodium arsenite (1.0 microM) increases their proliferation, causes DNA double-strand breaks, and induce anchorage-independent <em>growth</em>. In arsenite-exposed cells, the levels of phospho-p53, p<em>2</em>1, and mdm<em>2</em> increase at early times after exposure. The levels, however, decrease with longer times. Interaction of the promoter of mot-<em>2</em> (a p53 inhibitor) with nuclear <em>factor</em> kappaB (NF-kappaB) was established by Southwestern and Western blot assays. Blockage of NF-kappaB prevents the increases of arsenite-induced mot-<em>2</em> levels, and knockdown of mot-<em>2</em> facilitates the nuclear translocation of p53, indicating that, in HaCaT cells exposed to arsenite, NF-kappaB inhibits p53 function by mot-<em>2</em>. Moreover, inactivation of NF-kappaB facilitated p53-mediated DNA repair and prevented arsenite-induced malignant transformation. Together, the results suggest that the repressive effect of NF-kappaB on p53 by mot-<em>2</em> leads to genomic instability, which is involved in arsenite-induced malignant transformation of human <em>keratinocytes</em>.

We studied the effects of serine proteases on cytokine gene expression by cultured normal human <em>keratinocytes</em>. In resting <em>keratinocytes</em>, steady-state mRNA levels for interleukins IL-1 alpha, IL-1 beta, IL-7, and IL-8, transforming <em>growth</em> <em>factors</em> alpha and beta, and tumor necrosis alpha were sufficient to be detected by our reverse transcriptase-polymerase clozin reaction method. Incubation of <em>keratinocytes</em> with <em>2</em>5 nM trypsin or 1 unit/ml thrombin for <em>2</em>4 hr selectively upregulated mRNA levels for granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) and Il-6 to detectable levels. <em>Keratinocytes</em> secreted GM-CSF and IL-6 protein in response to these proteases. Monensin did not inhibit the gene expression for the cytokines, thereby excluding the possibility of intervention by secreted molecules. Aprotinin and argatroban inhibited the effects of the proteases. SFLLRN and SLIGRL, tethered ligand receptor peptides for thrombin receptor and for proteinase-activated receptor <em>2</em> (PAR-<em>2</em>), respectively, duplicated the effects of the proteases on <em>keratinocytes</em>, which expressed mRNA for both receptors. Trypsin increased tyrosine phosphorylated proteins and intracellular free calcium concentrations. Tyrphostin, pertussis toxin, or H-7 suppressed trypsin- and thrombin-induced GM-CSF gene expression. Our results demonstrate that the serine proteases activate thrombin receptors and PAR-<em>2</em> on <em>keratinocytes</em>, triggering intracellular signaling and then inducing the synthesis of GM-CSF. We speculate that serine proteases modulate the course of physiological and pathological processes in the skin by stimulating <em>keratinocytes</em> to produce the cytokines.

OBJECTIVE

Pancreatic ductal adenocarcinoma is a deadly disease because of late diagnosis and chemoresistance. We aimed to find a panel of serum cytokines representing diagnostic and predictive biomarkers for pancreatic cancer.

METHODS

A cytokine antibody array was performed to simultaneously identify 507 cytokines in sera of patients with pancreatic cancer and healthy controls. The nonparametric Mann-Whitney U test was used to pairwise compare the controls, the pretreated patients, and the posttreated patients. Fold changes greater than or equal to 1.5 or less than or equal to 1/1.5 were considered significant. Receiver operating characteristic curves were used to assess the performance of the model. A leave-one-out cross-validation was used for estimating prediction error.

RESULTS

Comparing the sera of pretreated patients against the control samples, the cytokines fibroblast <em>growth</em> <em>factor</em> 10 (FGF-10/<em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), chemokine (C-X-C motif) ligand 11 interferon inducible T cell alpha chemokine (I-TAC)/chemokine [C-X-C motif] ligand 11 (CXCL11), oncostatin M (OSM), osteoactivin/glycoprotein nonmetastatic melanoma protein B, and stem cell <em>factor</em> (SCF) were found significantly overexpressed. Besides, the cytokines CD30 ligand/tumor necrosis <em>factor</em> superfamily, member 8 (TNFSF8), chordin-like <em>2</em>, FGF-10/KGF-<em>2</em>, <em>growth</em>/differentiation <em>factor</em> 15, I-TAC/CXCL11, OSM, and SCF were differentially expressed in response to treatment.

CONCLUSIONS

We propose a role for FGF-10/KGF-<em>2</em>, I-TAC/CXCL11, OSM, osteoactivin/glycoprotein nonmetastatic melanoma protein B, and SCF as novel diagnostic biomarkers. CD30 ligand/TNFSF8, chordin-like <em>2</em>, FGF-10/KGF-<em>2</em>, <em>growth</em>/differentiation <em>factor</em> 15, I-TAC/CXCL11, OSM, and SCF might represent as predictive biomarkers for gemcitabine and erlotinib response of patients with pancreatic cancer.

Hypoxia limits wound healing. Both normobaric (1 atm) and hyperbaric oxygen (HBO) approaches have been used clinically to oxygenate wound tissue. Recently, we reported that HBO ameliorates stress-induced impairment of dermal healing. We examined the effect of pressure on oxygen-induced vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression by human HaCaT <em>keratinocytes</em>. Next, we investigated the effect of HBO on whole-body redox and on the ratio of oxidized to reduced glutathione (GSSG/GSH) in the liver, heart, lung, and brain of rats. Superoxygenation (90% O<em>2</em>) of <em>keratinocytes</em> partially arrested cell <em>growth</em>. G<em>2</em>-M <em>growth</em> arrest was substantially augmented by HBO. HBO also caused apoptosis in a small subpopulation. Normobaric oxygen, but not HBO (<em>2</em> atm), potently induced the expression of VEGF165 and 189. In vivo electron paramagnetic resonance spectroscopy imaging revealed a clear shift of the whole-body redox status toward oxidation in response to HBO. The standard diet of laboratory rats contains excessive (17x human recommended dietary allowance) alpha-tocopherol (E++), which confers exceptional resistance to oxidant insults. People with chronic wounds commonly suffer from under- or malnutrition. We generated vitamin E-deficient (E-) rats by long-term dietary vitamin E restriction. HBO did not raise GSSG/GSH in E++ rats, but post-HBO GSSG/GSH was significantly higher in E- compared with E++. Thus, rats on antioxidant-enriched diet were well protected against HBO. The risk of oxidative stress may negatively impact the net benefits of HBO. This is of special concern for people with inadequate intake of dietary antioxidants. Nutritional antioxidant supplementation may offset HBO-induced oxidative stress.

Fibroblast <em>growth</em> <em>factor</em> 10 is a novel member of the fibroblast <em>growth</em> <em>factor</em> family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (or fibroblast <em>growth</em> <em>factor</em> 7), a key mediator of <em>keratinocyte</em> <em>growth</em> and differentiation. Both fibroblast <em>growth</em> <em>factor</em> 10 and <em>keratinocyte</em> <em>growth</em> <em>factor</em> bind with high affinity to the tyrosine kinase <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor. Here we analyzed the effect of fibroblast <em>growth</em> <em>factor</em> 10 on primary cultures of human <em>keratinocytes</em>, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast <em>growth</em> <em>factor</em> 10 with those induced by <em>keratinocyte</em> <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em>. Cell counting, 5-bromo-<em>2</em>'-deoxyuridine incorporation, and western blot analysis showed that fibroblast <em>growth</em> <em>factor</em> 10, similarly to <em>keratinocyte</em> <em>growth</em> <em>factor</em>, not only is a potent mitogen for human <em>keratinocytes</em>, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca<em>2</em>+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast <em>growth</em> <em>factor</em> 10, similarly to <em>keratinocyte</em> <em>growth</em> <em>factor</em>, is able to induce tyrosine phosphorylation of <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor and of cellular substrates such as PLCgamma.

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin <em>keratinocytes</em>, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming <em>growth</em> <em>factor</em>-β (TGF-β) signaling by protecting phospho-Smad <em>2</em> and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits <em>growth</em>. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor <em>growth</em>, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor <em>growth</em> and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the <em>growth</em> of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.

Interleukin (IL)-<em>2</em>4 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-<em>2</em>4 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-<em>2</em>4 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-<em>2</em>4 is expressed in <em>keratinocytes</em> during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days <em>2</em>, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-<em>2</em>4. Protein expression was detected in the <em>keratinocyte</em> population with maximum expression at days <em>2</em> and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming <em>growth</em> <em>factor</em> alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-<em>2</em>4 protein expression in normal human epidermal <em>keratinocytes</em> (NHEKs). Examination of the function of IL-<em>2</em>4 in both in vitro wound repair and migration assays demonstrated that IL-<em>2</em>4 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-<em>2</em>4 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of <em>keratinocytes</em>.