Psoriasis is a common chronic inflammatory disease of the skin characterized by epidermal hyperplasia and angiogenesis. Recently, vascular endothelial <em>growth</em> <em>factor</em> receptors (VEGFRs, including VEGFR-1, VEGFR-<em>2</em> and VEGFR-3) were found to be expressed in normal human epidermis and associated with proliferation and migration of <em>keratinocytes</em>. The purpose of this study is to investigate the expression of VEGFRs on psoriatic <em>keratinocytes</em> and the roles of calcium and VEGF in regulating VEGFR expression. Skin samples from 17 patients with chronic plaque psoriasis and 11 normal controls were included. The expression of VEGFRs in psoriatic <em>keratinocytes</em> at mRNA and protein levels was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Localization of the VEGFRs in skin lesions was determined by immuno-fluorescent method. Since <em>keratinocyte</em> proliferation and differentiation rely on calcium concentrations, and VEGF is overexpressed in psoriatic epidermis, we further investigated the roles of calcium and VEGF in regulating the expression of VEGFRs. Overexpression of VEGFR-1, VEGFR-<em>2</em> and VEGFR-3 in psoriatic epidermis was demonstrated both at mRNA and protein levels in vitro. VEGFRs were strongly labeled in non-lesional, perilesional and lesional psoriatic <em>keratinocytes</em> in all viable epidermal stratums in vivo. Furthermore, both exogenous VEGF165 and calcium enhanced the expression of VEGFRs. Calcium also enhanced the expression of VEGF in non-lesional psoriatic <em>keratinocytes</em>, while targeted blockade of VEGF activity by bevacizumab could not inhibit calcium-induced up-regulation of protein levels of VEGFRs. We conclude from these results that VEGFRs are overexpressed in lesional psoriatic epidermal <em>keratinocytes</em>. Both calcium and VEGF regulate VEGFRs expression in psoriatic epidermis. More importantly, calcium is a potential regulator for VEGFR independent of VEGF.

Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal <em>growth</em> <em>factor</em> receptors in human <em>keratinocytes</em> produces 15% to <em>2</em>00% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several <em>factors</em> including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta <em>2</em>-adrenergic receptors, isoproterenol produces a <em>2</em>0-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 1<em>2</em> min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.

BACKGROUND

Hepatocyte growth factor (HGF) (also known as scatter factor (SF)) is a heterodimeric protein that is the most potent known complete mitogen for hepatocytes in culture. HGF is a mitogen for many epithelial cells including hepatocytes, kidney tubular epithelial cells, mammary epithelial cells, keratinocytes, etc. The protein encoded by the proto-oncogene c-met is the high affinity receptor for HGF. HGF concentration in the plasma dramatically increases after partial hepatectomy and in fulminant hepatic failure. This study describes the pharmacokinetics of HGF in the rat.

METHODS

Human recombinant HGF (a gift from Genentech) was radioiodinated and shown to retain biologic activity and structure. Approximately 74 ng of [125I]HGF was injected into the penile vein of male Fisher rats 5 minutes after a complete bile fistula and jugular venous catheterization were performed for blood and bile sampling. Half of the rats were subjected to 70% partial hepatectomy.

RESULTS

The percentage of injected radioactivity present in the liver of control rats was 29.5% +/- 0.5% at 15 minutes and decreased to 8.6% +/- 1.0% at 120 minutes; the kidneys had 6.2% +/- 0.2% at 15 minutes, decreasing to 1.48% +/- 0.3% at 120 minutes. All the other organs examined had less than 1% of the injected radioactivity. The remaining radioactivity was present in low affinity sites in blood, bone, muscle, and skin. In control rats, radioactivity appeared in the bile within 3 minutes, reached a peak between 40 to 50 minutes, and tapered thereafter for a total 2-hour collection of 2.3% +/- 0.5%. In the partially hepatectomized rats, the HGF blood clearance was decreased (partial hepatectomy = 0.27 +/- 0.03 ml/minute; control = 0.53 +/- 0.06 ml/minute, p < 0.006), and the terminal half-life prolonged (partial hepatectomy = 124 +/- 11 minutes; control = 83 +/- 10 minutes, p < 0.03). The initial half-life for HGF, as extrapolated from the chart, was estimated at 3.8 minutes in control rats.

CONCLUSIONS

Liver is the principle organ for initial uptake of [125I]HGF; disappearance from the blood suggests multicompartment kinetics with a rapid phase and a slower phase; only a portion of the hepatic uptake appears in the bile; and partial hepatectomy decreases the blood clearance of [125I]HGF. These results are correlated with previous findings bearing on the role of HGF elevation after partial hepatectomy as a stimulus for transfer of hepatocytes from G0 to G1 early in liver regeneration after partial hepatectomy.

We have recently shown that UVB radiation activates epidermal <em>growth</em> <em>factor</em> receptor (EGFR)/extracellular regulated kinase 1 and <em>2</em> (ERK1/<em>2</em>) and p38 signaling pathways in <em>keratinocytes</em>. However, the functional relevance of these processes for downstream signaling and cell survival remains to be determined. The specific EGFR inhibitor PD153035 markedly decreased UVB-induced phosphorylation of EGFR, ERK1/<em>2</em> and shc, whereas p38 activation was unaffected. PD153035 pretreatment followed by UVB reduced clonogenic potential and enhanced peroxide production, apoptosis and cell death. Our data suggest that ligand-independent phosphorylation of EGFR and likely dependent downstream signaling pathways regulate cellular defense mechanisms important for cell survival following oxidative stress.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>/fibroblast <em>growth</em> <em>factor</em> 7 (KGF/FGF7) is known to be a potent <em>growth</em> <em>factor</em> for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related <em>growth</em> <em>factor</em> FGF10. We also determined the <em>factors</em> that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells. Using an FGF7-specific antibody which does not react with the FGF7 heparan sulphate proteoglycan (HSPG)-binding site, we showed epithelial and myoepithelial immunohistochemical staining in normal breast sections, and epithelial staining in breast carcinomas. Stromal staining was also detected in some lobular carcinomas as well as a subset of invasive ductal carcinomas. FGF10 and FGF receptor (FGFR)<em>2</em> immunostaining showed a similar epithelial expression pattern, whereas no stromal staining was observed. We purified normal breast stromal, epithelial and myoepithelial cells and showed that FGF7 stimulated proliferation of both epithelial cell types, but not stromal fibroblasts. We also examined the effects of FGF7 on Matrigel-embedded organoids, containing both epithelial and myoepithelial cells, and showed FGF7 induced an increase in cellular proliferation. Furthermore, conditioned medium derived from stromal cells was shown to increase the proliferation of normal and neoplastic breast epithelial cells, which could be abolished by a neutralising antibody to FGF7. Finally, we showed that interleukin-1beta, but not oestradiol or other oestrogen receptor ligands, caused a dose-related FGF7 release. Further results also indicate that the epithelial localisation of FGF7 and FGF10 in breast tissue sections is likely to be due to their binding to their cognate receptor. In summary, our findings suggest that FGF7 is a paracrine <em>growth</em> <em>factor</em> in the breast. FGF7 is produced by the breast stromal fibroblasts and has profound proliferative and morphogenic roles on both epithelial and myoepithelial cells.

In this study, we examined the effects of dexamethasone (DEX) on airway branching and subsequent lung maturation. DEX treatment of fetal rat lung explants was initiated during the early pseudoglandular stage of development. Day 14 fetal lung explants were cultured with and without DEX for 4 d. Explants treated with 10 nM or higher concentrations of DEX showed features of both distorted and accelerated maturation. DEX-treated lungs had <em>growth</em> retardation, distorted branching, dilated proximal tubules, and suppressed proliferation of epithelial cells of the distal tubules. Several biochemical and morphologic features of accelerated maturation were also observed: 1) the epithelial cells lining the distal tubules (prospective respiratory airways) were generally cuboidal or flattened; <em>2</em>) the cuboidal cells often contained lamellar bodies and abundant glycogen; 3) rudimentary septa and large airspace were present; 4) mesenchymal tissue was attenuated and compressed between adjacent epithelial tubules; 5) the distribution of SP-C mRNA in distal tubules was more mature, with individual and clusters of cells expressing SP-C transcripts; and 6) the transcript levels of several genes related to epithelial <em>growth</em> [<em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), KGF receptor, and hepatocyte <em>growth</em> <em>factor</em> receptor] and differentiation [surfactant proteins, SP-A, SP-B and SP-C and the Clara cell secretory protein, CC10] were precociously increased. These results show that DEX treatment of the lung during the early pseudoglandular stage accelerates the acquisition of several features of advanced maturation that normally accompany late stages of fetal development. We postulate that KGF mediates at least some effects of DEX on lung maturation and gene expression.

Radiation-induced gastrointestinal toxicity is due in part to the killing of the clonogenic crypt cells and eventual depopulation of the villi. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), a member of the fibroblast <em>growth</em> <em>factor</em> family (FGF-7), has been shown to stimulate proliferation of cells along the murine digestive tract from the foregut to the colon. Using an in vivo microcolony assay, we found that 1.0 mg/kg KGF administered intravenously (i.v.) for 3 consecutive days (<em>2</em> days before, 1 day before and <em>2</em> h after irradiation) increased the number of surviving crypts by a <em>factor</em> of <em>2</em>.6, <em>2</em>.7 and <em>2</em>.4 in the duodenum, jejunum and ileum, respectively, after a single-dose whole-body irradiation (10-16 Gy) (P < 0.001). Treatment of mice with KGF i.v. significantly increased the D0 of the radiation survival curves by 0.37, 0.<em>2</em><em>2</em> and 0.36 Gy, leading to dose modification <em>factors</em> of 1.<em>2</em>8, 1.16 and 1.<em>2</em>4 for duodenal, jejunal and ileal crypt cells, respectively. Similar results were obtained with KGF administered subcutaneously. Treatment with both KGF and stem cell <em>factor</em> (previously shown to enhance intestinal crypt survival after total-body irradiation) increased the number of surviving crypt cells after irradiation to levels similar to that in animals treated with KGF alone. Administration of KGF for 7 consecutive days (beginning <em>2</em> days prior to irradiation) increased the LD(50/10) from 5.50 Gy/day to 5.90 Gy/day (P = 0.05) for animals irradiated with five daily fractions to a local abdominal field. These results suggest that KGF may be of clinical value in reducing radiation toxicity to the intestine.

We examined possible roles of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) in lung morphogenesis. By polymerase chain reaction, transcripts for both KGF and its receptor were detected early (rat gestational days 16 and 14, respectively) and their abundance increased during lung morphogenesis. To evaluate possible role of KGF in lung morphogenesis, day 14 lung explants were cultured in Dulbecco's modified Eagle medium + 10% fetal calf serum for 1 to 4 days in the presence (5-50 ng/ml) or absence of KGF (control). KGF (at <em>2</em>5 and 50 ng/ml) induced a marked reduction in the number of terminal branches and destination of the distal epithelium into cyst-like structures. These effects of exogenous KGF were progressively diminished by increasing concentrations of anti-KGF (<em>2</em>-16 micrograms/ml). Electron microscopic examination revealed that the epithelial cells of the cystic structures contained lamellar bodies, and were therefore type II cells and/or their progenitors. Northern blot analysis showed higher expression of surfactant protein C (SP-C) mRNA (a marker for alveolar epithelial type II cells) in KGF-treated fetal lungs. In situ hybridization of the KGF-treated lungs revealed that the SP-C mRNA-expressing cells were arranged distally in the form of linear arrays, a pattern distinctly different from that in control lungs. Acidic fibroblast <em>growth</em> <em>factor</em>, which also binds KGF receptors, in the presence of heparin mimicked the effect of KGF on branching. Transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta 1) inhibited branching of fetal lungs in culture, and this effect dominated over that induced by KGF. Blocking of endogenous HGF with antibodies or addition of HGF to cultures of fetal lung explants had no significant effect on branching or <em>growth</em>. In conclusion, KGF markedly influences branching, and epithelial <em>growth</em>, differentiation, and patterning during lung morphogenesis.

p1<em>2</em>(CDK<em>2</em>-AP1) (p1<em>2</em>) is a <em>growth</em> suppressor isolated from normal <em>keratinocytes</em>. Ectopic expression of p1<em>2</em> in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p1<em>2</em> to bind to both DNA polymerase alpha/primase and to cyclin-dependent kinase <em>2</em> (CDK<em>2</em>), thereby inhibiting their activities. We report in this article that in normal epithelial cells, transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1) induces p1<em>2</em> expression transcriptionally, which, in turn, mediates the <em>growth</em> inhibitory activity of TGF-beta1. We created inducible p1<em>2</em> antisense HaCaT cell lines [ip1<em>2</em> (-) HaCaT] and showed that selective reduction of cellular p1<em>2</em> resulted in an increase in: (a) CDK<em>2</em>-associated kinase activity; (b) protein retinoblastoma (pRB) phosphorylation; and (c) [(3)H]thymidine incorporation, and partially reversed TGF-beta1-mediated inhibition of CDK<em>2</em> kinase activity, pRB phosphorylation, and cell proliferation. Furthermore, we generated p1<em>2</em>-deficient mouse oral <em>keratinocytes</em> (MOK(p1<em>2</em>-/-)) and compared their <em>growth</em> characteristics and response to TGF-beta1 with that of wild-type mouse oral <em>keratinocytes</em> (MOK(WT)). Under normal culture conditions, the number of MOK(p1<em>2</em>-/-) in S phase is <em>2</em>-fold greater than that of MOK(WT). Concomitantly, fewer cells are in G(<em>2</em>) phase in MOK(p1<em>2</em>-/-) than that in MOK(WT). Moreover, response to TGF-beta1-mediated <em>growth</em> suppression is compromised in MOK(p1<em>2</em>-/-) cells. Mechanistic studies showed that MOK(p1<em>2</em>-/-) have increased CDK<em>2</em> activity and reduced sensitivity to inhibition by TGF-beta1. Collectively our data suggest that p1<em>2</em> plays a role in TGF-beta1-mediated <em>growth</em> suppression by modulating CDK<em>2</em> activities and pRB phosphorylation.

Melanoma <em>growth</em>, angiogenesis and metastatic progression are strongly promoted by the inflammatory tumor microenvironment due to high levels of cytokine and chemokine secretion by the recruited inflammatory and stromal cells. In addition, platelets and molecular components of procoagulant pathways have been recently emerging as critical players of tumor <em>growth</em> and metastasis. In particular, thrombin, through the activity of its receptor protease-activated receptor-1 (PAR-1), regulates tumor cell adhesion to platelets and endothelial cells, stimulates tumor angiogenesis, and promotes tumor <em>growth</em> and metastasis. Notably, in many tumor types including melanoma, PAR-1 expression directly correlates with their metastatic phenotype and is directly responsible for the expression of interleukin-8, matrix metalloproteinase-<em>2</em> (MMP-<em>2</em>), vascular endothelial <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, and integrins. Another proinflammatory receptor-ligand pair, platelet-activating <em>factor</em> (PAF) and its receptor (PAFR), have been shown to act as important modulators of tumor cell adhesion to endothelial cells, angiogenesis, tumor <em>growth</em> and metastasis. PAF is a bioactive lipid produced by a variety of cells from membrane glycerophospholipids in the same reaction that releases arachidonic acid, and can be secreted by platelets, inflammatory cells, <em>keratinocytes</em> and endothelial cells. We have demonstrated that in metastatic melanoma cells, PAF stimulates the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and activating transcription <em>factor</em> 1 (ATF-1), which results in overexpression of MMP-<em>2</em> and membrane type 1-MMP (membrane type 1-MMP). Since only metastatic melanoma cells overexpress CREB/ATF-1, we propose that metastatic melanoma cells are better equipped than their non-metastatic counterparts to respond to PAF within the tumor microenvironment. The evidence supporting the hypothesis that the two G-protein coupled receptors, PAR-1 and PAFR, contribute to the acquisition of the metastatic phenotype of melanoma is presented and discussed.

The immunomodulatory cationic host defence peptide LL-37 plays an important role in epithelial innate immunity; at higher concentrations (<em>2</em>0-50 microg mL(-1)) associated with inflammation, LL-37 elicits the production of cytokines and chemokines. It was demonstrated here that lower, physiologically relevant LL-37 concentrations (<em>2</em>-3 microg mL(-1)) altered epithelial cell responses to proinflammatory stimuli. In combination with interleukin-1beta (IL-1beta) and the Toll-like receptor-5 (TLR5) agonist flagellin, these low concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated <em>keratinocytes</em> and by bronchial epithelial cells. In combination with the TLR<em>2</em>/1 agonist PAM3CSK4, LL-37 synergistically induced transcription and the release of both IL-8 and IL-6 from primary bronchial epithelial cells; the IL-8 response was demonstrated to be regulated by epidermal <em>growth</em> <em>factor</em> receptor signalling. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist polyI:C resulted in synergistic increases in IL-8 release and cytotoxicity. These data indicate that low concentrations of LL-37 may alter epithelial responses to infecting microorganisms in vivo.

The protein C (PC) pathway plays an important role in coagulation and inflammation. Many components of the PC pathway have been identified in epidermal <em>keratinocytes</em>, including endothelial protein C receptor (EPCR), which is the specific receptor for PC/activated PC (APC), but the core member of this pathway, PC, and its function in <em>keratinocytes</em> has not been defined. In this study, we reveal that PC is strongly expressed by human <em>keratinocytes</em> at both gene and protein levels. When endogenous PC was blocked by siRNA the proliferation of <em>keratinocytes</em> was significantly decreased. This inhibitory effect was restored by the addition of recombinant APC. PC siRNA treatment also increased cell apoptosis by 3-fold and inhibited cell migration by more than <em>2</em>0%. When <em>keratinocytes</em> were pretreated with RCR<em>2</em>5<em>2</em>, an EPCR-blocking antibody, or PD153035, an epidermal <em>growth</em> <em>factor</em> receptor (EGFR) inhibitor, cell proliferation was hindered by more than 30%. These inhibitors also completely abolished recombinant APC (10 mug/ml)-stimulated proliferation. Blocking PC expression or inhibiting its binding to EPCR/EGFR decreased the phosphorylation of ERK1/<em>2</em> but increased p38 activation. Furthermore, inhibition of ERK decreased cell proliferation by approximately 30% and completely abolished the stimulatory effect of APC on proliferation. Taken together, these results indicate that <em>keratinocyte</em>-derived PC promotes cell survival, <em>growth</em>, and migration in an autocrine manner via EPCR, EGFR, and activation of ERK1/<em>2</em>. Our results highlight a novel role for the PC pathway in normal skin physiology and wound healing.

Human epidermal <em>keratinocytes</em> were established in culture using a low-Ca<em>2</em>+ (0.15 mM), serum-free <em>keratinocyte</em> <em>growth</em> medium (KGM) as the culture medium. Early passage <em>keratinocytes</em> (i.e., between passages 3-8) were incubated for 1 or <em>2</em> d in KGM, in KGM supplemented with 1.4 mM Ca<em>2</em>+, or in <em>growth</em> <em>factor</em>-deprived <em>keratinocyte</em> basal medium (KBM). The cells were concomitantly treated with all-trans retinoic acid (0.1-<em>2</em>.5 micrograms/ml), and cell <em>growth</em> was quantitated at the end of the incubation period. The <em>keratinocytes</em> were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN). Treatment with all-trans retinoic acid inhibited proliferation of <em>keratinocytes</em> that were rapidly <em>growing</em> in KGM. Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca<em>2</em>+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca<em>2</em>+. In contrast to these effects, all-trans retinoic acid treatment of <em>keratinocytes</em> in KBM, in which the cells were normally quiescent, stimulated <em>growth</em>. In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of <em>keratinocyte</em> proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate). <em>Keratinocyte</em> adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions. In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid. FN production was also inhibited but to a lesser degree. Concentrations of all-trans retinoic acid required to maximally inhibit <em>keratinocyte</em> adhesion and matrix production were higher (1.0-<em>2</em>.5 microgram/ml) than the concentration required to stimulate proliferation in KBM. These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced <em>keratinocyte</em> proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.

Normal adult human <em>keratinocytes</em> in monolayer culture and SCL-1, a skin-derived squamous-cell carcinoma cell line, were investigated for the expression of receptors for insulin-like <em>growth</em> <em>factors</em> (IGF) and insulin. As demonstrated by affinity crosslinking, radiolabeled IGF-1, IGF-<em>2</em>, and insulin bound specifically to both cell types. Each cell expressed type I IGF receptors, with affinity for IGF-1 greater than IGF-<em>2</em> much greater than insulin. Insulin receptors, with highest affinity for insulin, were also present on both cells. However, <em>keratinocytes</em> and SCL-1 cells differed in 1<em>2</em>5I-IGF-<em>2</em> binding. 1<em>2</em>5I-IGF-<em>2</em>-bound to both type I and type II IGF receptors in normal <em>keratinocytes</em>, but bound predominantly to membrane-associated IGF binding proteins in SCL-1. IGF-1 was slightly more potent than IGF-<em>2</em> in stimulating <em>growth</em> of both <em>keratinocytes</em> and SCL-1 cells. In <em>keratinocytes</em>, concentrations of IGF-1 ranging from 5-100 ng/ml, and of IGF-<em>2</em> from 50-100 ng/ml, resulted in a significant increase in cell number. At the maximum dose of 100 ng/ml, either IGF-1 or IGF-<em>2</em> caused a <em>2</em>.3-times increase in cell number. In SCL-1 cells, IGF-1 was more potent than IGF-<em>2</em> or insulin at lower concentrations, but either IGF-1 or IGF-<em>2</em> at the maximal concentration of 333 ng/ml stimulated a 4.7-times increase in thymidine incorporation. The stimulatory effect of insulin in SCL-1 was 10-50 times less potent than that of the IGF. The effect of either IGF on SCL-1 was completely inhibited by the type I IGF receptor antibody alpha IR-3, suggesting that both IGFs are mitogenic through the type I IGF receptor. Insulin action was partially blocked by alpha IR-3, suggesting that insulin can act through both the insulin and type I IGF receptors. It thus appears that IGF-1 and IGF-<em>2</em> are mitogens for normal and transformed human <em>keratinocytes</em> and that their actions are primarily mediated through the type I IGF receptor, whereas insulin is a mitogen through both the IGF-1 receptor and the insulin receptor.

<em>2</em>,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent modulator of epithelial cell <em>growth</em> and differentiation, has been shown to induce transforming <em>growth</em> <em>factor</em> (TGF)-alpha in cultures of human <em>keratinocytes</em> and in the human <em>keratinocyte</em> cell line, SCC-1<em>2</em>F. In this report, we investigated the mechanisms by which TCDD alters TGF-alpha expression. In addition, we studied the actions of TCDD on TGF-beta 1 and TGF-beta <em>2</em> expression. Treatment of SCC-1<em>2</em>F cells with TCDD resulted in an increase in TGF-alpha and a reduction in TGF-beta <em>2</em> mRNA levels while mRNA levels for TGF-beta 1 were unchanged. Changes in TGF-alpha and TGF-beta <em>2</em> expression were maximal by <em>2</em>4 h. No change in the rate of transcription of TGF-alpha was detected following treatment with TCDD as determined by nuclear run-off analysis. TCDD treatment resulted in a stabilization of TGF-alpha mRNA as judged by an approximately <em>2</em>-fold higher level of TGF-alpha mRNA in treated versus control cells in the presence of actinomycin D. In contrast to TGF-alpha, the rate of transcription of TGF-beta <em>2</em> was significantly reduced following TCDD treatment. These findings demonstrate that the induction of TGF-alpha expression in SCC-1<em>2</em>F cells by TCDD occurs post-transcriptionally, primarily by mRNA stabilization, while TGF-beta <em>2</em> expression is reduced due to a decrease in the rate of TGF-beta <em>2</em> gene transcription.

Hyaluronic acid (HA), a glycosaminoglycan located between <em>keratinocytes</em> in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat <em>keratinocyte</em> organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to <em>2</em>4 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by <em>2</em>4 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive <em>factor</em>. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble <em>factor</em>. From earlier evidence, we hypothesized that an EGF-like <em>growth</em> <em>factor</em> might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (<em>2</em>) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) blocked wounding-induced HA synthesis by>> or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptor VEGFR-<em>2</em> play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal <em>keratinocytes</em>. Emerging data suggest that <em>keratinocyte</em>-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that <em>keratinocytes</em> from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and <em>2</em>). To define the functional significance of VEGFR-<em>2</em> in epidermis, we examined its role in a <em>keratinocyte</em> cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-<em>2</em> on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF165 treatment. Treatment of HaCaT cells with VEGF165 induced tyrosine-autophosphorylation of VEGFR-<em>2</em> and phosphorylation of PLC-gamma and p44/4<em>2</em> MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-<em>2</em> (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF165 stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-<em>2</em> in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-<em>2</em> expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.

Basic fibroblast <em>growth</em> <em>factor</em> (bFGF), but not acidic fibroblast <em>growth</em> <em>factor</em> (aFGF), was found to be mitogenic for cultured mouse <em>keratinocytes</em>. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to <em>keratinocytes</em> arrested in serum-free and <em>growth</em> <em>factor</em>-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.<em>2</em> and 0.7 ng/ml. In the same culture system, insulin-like <em>growth</em> <em>factor</em> I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with <em>2</em>-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse <em>keratinocytes</em>. In this regard they are comparable to epidermal <em>growth</em> <em>factor</em>, which shows a half-maximal stimulation at a concentration between 1.5-<em>2</em> ng/ml. These results show that in addition to mesenchymal cells, FGF is a <em>growth</em> <em>factor</em> not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the <em>growth</em> promoting effect of insulin on <em>keratinocytes</em> may be mediated by the IGF-I receptor.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) administered by intratracheal instillation is well documented to stimulate the proliferation of alveolar and bronchial cells. In the present study, intravenous KGF was also shown to stimulate the proliferation of alveolar and bronchial cells in mice and rats, although to a lesser degree than intratracheal KGF. Despite the decreased potency of intravenous KGF on pulmonary cell 5-bromo-<em>2</em>'-deoxyuridine incorporation compared with intratracheal KGF, intravenous KGF was very effective in preventing experimental bleomycin-induced pulmonary dysfunction, weight loss, and mortality in either mice or rats and experimental hyperoxia-induced mortality in mice. The effectiveness of intravenous administration of KGF in preventing lung injury suggests that the mechanisms of the protective effect of KGF may involve more than pulmonary cell proliferation and also suggests the potential use of systemic KGF for clinical trials in settings of pulmonary injury.

Inflammatory bowel disease (IBD) affects millions of people worldwide but its pathophysiology remains unclear. Therefore, experimental models of colitis have contributed crucially for the understanding of IBD, and also in the investigations for effective therapies. Herein we investigated the kinetics of inflammatory mediator production and cell infiltration during acute and chronic dextran sodium sulfate (DSS)-induced colitis. The induction phases with DSS were characterized by severe disease activity with massive colonic polymorphonuclear infiltration and increased levels of tumor necrosis <em>factor</em>-α (TNF-α), <em>keratinocyte</em>-derived chemokine (CXCL1/KC), interleukin (IL)-17 and vascular adhesion molecule-1 (VCAM-1). Interestingly, in the recovery periods, we found marked increase of anti-inflammatory mediators IL-10, IL-4, transforming <em>growth</em> <em>factor</em>-β (TGF-β) and cyclooxygenase <em>2</em> (COX-<em>2</em>) that seems be essential for the resolution of intestinal inflammation. Furthermore, nuclear <em>factor</em> κB (NFκB) and regulatory T cell marker forkhead box P3 (FoxP3) were increased gradually during experimental colitis, demonstrating a discrepant profile response and evident immune disbalance in the chronic phase of intestinal mucosal inflammation. Taken together, these results provide valuable information for studies on DSS-induced colitis and especially for the identification of biomarkers that predict disease course and possible therapeutic interventions.

Biologically active nerve <em>growth</em> <em>factor</em> (NGF) is synthesised and released by proliferating normal human <em>keratinocytes</em>. NGF up-regulates the expression of NGF mRNA in <em>keratinocytes</em>. <em>Keratinocytes</em> express both the low (p75)- and the high-affinity (TrkA) NGF-receptors, which are located in the basal layer of the epidermis. K<em>2</em>5<em>2</em>, a specific inhibitor of trk phosphorylation, blocks NGF-induced <em>keratinocyte</em> proliferation, in absence of exogenous NGF. Normal <em>keratinocytes</em> over-expressing TrkA proliferate better than control transfectants, while the NGF mimicking anti-Trk antibody induces an increased <em>keratinocyte</em> proliferation in Trk over-expressing cells as compared to mock transfected <em>keratinocytes</em>. In addition, NGF over-expressing <em>keratinocytes</em> proliferate better than mock transfected cells. K<em>2</em>5<em>2</em>, by blocking TrkA phosphorylation, induces apoptosis in normal <em>keratinocytes</em>, but not in <em>keratinocytes</em> over-expressing bcl-<em>2</em>. Furthermore, NGF transfected <em>keratinocytes</em> are protected from UV-B-induced <em>keratinocyte</em> apoptosis, by maintaining constant levels of Bcl-<em>2</em> and Bcl-xL . Taken together these results support the concept of an autocrine survival system sustained by NGF and its high-affinity receptor in human <em>keratinocytes</em>. Because NGF and Trk levels are highly expressed in psoriasis. one could speculate that NGF autocrine system plays a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer.

OBJECTIVE

The aim of this study was to develop an in vitro human gastric stem and/or progenitor cell model that may be used to study the mechanism of gastric carcinogenesis induced by Helicobacter pylori infection.

METHODS

Human gastric biopsy was minced and digested with collagenase and dispase and cultured in a low-calcium medium (serum-free <em>keratinocyte</em> medium; <em>keratinocyte</em>-SFM) supplemented with N-acetyl-L-cysteine and L-ascorbic acid <em>2</em>-phosphate. Actively proliferating epithelial colonies with sustained <em>growth</em> were isolated and characterized for karyotype and phenotypes related to stem cell characteristics including proliferation and differentiation potential, ability of anchorage-independent <em>growth</em> (AIG), gap junctional intercellular communication (GJIC) and the expression of Oct-4, a transcription <em>factor</em> previously shown to be expressed in embryonic stem cells, adult stem cells and undifferentiated tumor cells. To study the carcinogenic effect of H. pylori infection, gastric stem and/or progenitor cells were incubated with H. pylori culture products and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, to see the telomerase activation.

RESULTS

Multiple cell lines with stem cell features were isolated by this new cell culture method. The results based on detailed characterization of one cell clone, KMU-GI<em>2</em>, revealed stem cell features of these cells. The initial clone contained mostly undifferentiated epithelial-like cells, which, upon subculture and propagation, gave rise to a heterogeneous cell population. Single cell-derived subclones, similar to the parental population, retained high differentiation potential and were capable of giving rise to many morphologically different cell types (i.e. epithelial-like, glial or neuron-like, round and various peculiar-shaped cells). Although these cells were normal in karyotype and competent in GJIC, they had the ability to grow in soft agar. Cells expressing epithelial membrane antigen (EMA), mucin 5AC, glial fibrillary acidic protein (GFAP), cytokeratin-18 (CK-18), trefoil <em>factor</em> 1 (TFF-1) and Oct-4 were found in the cell culture, but not E-cadherin-, gastrin- or telomerase-expressing cells. Furthermore, spontaneously immortalized non-tumorigenic clones could be derived from the cell population. After treating these cell cultures with the chemical carcinogen, MNNG and H. pylori culture products for 5 days, telomerase activity and telomerase mRNA expression were significantly elevated, while treatment with either of them showed no effect.

CONCLUSIONS

The new cell culture method can be used to develop gastric epithelial cell clones with sustained growth from endoscopic biopsy. The gastric cell clone showed several stem and/or progenitor cell phenotypes (i.e. the ability of AIG, high differentiation capacity, high susceptibility to spontaneous immortalization and the expression of Oct-4). The telomerase expression in these gastric stem and/or progenitor cells can be upregulated by exposure to H. pylori culture products and MNNG, an important step in neoplastic transformation. These results show that putative human gastric stem and/or progenitor cell clones can be developed by our method and these cells could be useful for studying the mechanisms of human gastric carcinogenesis including the mechanism of action of H. pylori, as well as the regulation of the proliferation and differentiation of human gastric mucosa.

The immediate appearance of platelets in wounds and the ability of platelets to release <em>growth</em> <em>factors</em> suggest that platelets are an important trigger of the tissue repair process. To examine the effect of systemic thrombocytopenia on both the inflammatory and proliferative aspects of wound healing, adult mice were rendered thrombocytopenic by intraperitoneal administration of a rabbit antimouse platelet serum. Full-thickness excisional dermal wounds were prepared and analyzed for inflammatory cell content, <em>growth</em> <em>factor</em> production, reepithelialization, collagen synthesis, and angiogenesis at multiple time points after injury. Compared to control mice, thrombocytopenic mice exhibited significantly altered wound inflammation. Wounds of thrombocytopenic mice contained significantly more macrophages and T cells, yet exhibited neutrophil content similar to wounds from control mice. Surprisingly, thrombocytopenic mice exhibited no delay in the reparative aspects of wound healing. The rate of wound reepithelialization, collagen synthesis, and angiogenesis was nearly identical for thrombocytopenic and control mice. Analysis of vascular endothelial <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factor</em> <em>2</em>, transforming <em>growth</em> <em>factor</em> beta1, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> revealed no difference in the levels of these <em>growth</em> <em>factors</em> in the wounds of control and thrombocytopenic mice. Taken together, the results suggest that the presence of platelets may influence wound inflammation, but that platelets do not significantly affect the proliferative aspects of repair, including wound closure, angiogenesis, and collagen synthesis.

The p63 gene, a member of the p53 family, is expressed as TA and ΔN isoforms. ΔNp63 is the predominant isoform expressed in cells of epithelial origin and frequently overexpressed in cancers. However, what regulates p63 expression is uncertain. Here, we showed that ΔNp63 is regulated by the transcription <em>factor</em> DEC1, a p53 family target. We also showed that the ability of DEC1 to regulate ΔNp63 is enhanced by histone deacetylase (HDAC) inhibitors or knockdown of histone deacetylase <em>2</em> (HDAC<em>2</em>). Consistent with this, we found that DEC1 and HDAC<em>2</em> physically interact and knockdown of HDAC<em>2</em> leads to increased binding of DEC1 to the ΔNp63 promoter. Interestingly, we found that <em>growth</em> suppression induced by HDAC inhibitors is attenuated by ectopic expression of DEC1 in a ΔNp63-dependent manner. In addition, we showed that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 promotes, <em>keratinocyte</em> differentiation via modulating ΔNp63 expression. Finally, we showed that DEC1 cooperates with HDAC inhibitors to further decrease <em>keratinocyte</em> differentiation. Together, we conclude that ΔNp63 is a novel target of DEC1 and HDAC<em>2</em> and modulates the efficacy of HDAC inhibitors in <em>growth</em> suppression and <em>keratinocyte</em> differentiation.