Alterations in gene expression represent key events in carcinogenesis. We have studied HPV-induced cervical carcinogenesis, using an HPV-33-positive cell line (UT-DEC-1) established from a low-grade vaginal dysplasia (VAIN-I). Early-passage cells contained HPV-33 in episomal form, but these were superseded at later passages by cells carrying only integrated virus. To gain insight into the biologic significance of HPV integration, we compared the level of gene expression in normal vaginal <em>keratinocytes</em>, early-passage and late-passage UT-DEC-1 cells, using cDNA microarrays. Total RNA was isolated from cells by CsCl-gradient centrifugation, reverse-transcribed with MMLV reverse transcriptase and labeled with alpha-(3<em>2</em>)P ATP. A cDNA microarray expression profile analysis was performed with Clontech's Human Cancer 1.<em>2</em> cDNA expression array kit. The 16 upregulated genes (cut-off <em>2</em>-fold), identified by comparing both cell types to control <em>keratinocytes</em>, appeared to support cell-cycle progression or to be functional in mitosis. These included, e.g., MCM4 DNA replication licensing <em>factor</em>, cdc<em>2</em>p34 and chromatin assembly <em>factor</em> 1 p48 subunit. Downregulated genes (44 altogether) interfered with apoptosis and cell adhesion, including the apoptosis-inducing genes FRAP, Bik and caspase-9 precursor. The most significant differences between the late and early passages (<em>2</em>9 and 46 constantly up- and downregulated genes without any fluctuation) were overexpression of the transcription <em>factors</em> E<em>2</em>F5 with its dimerization partner DP1, NF-kappa B and serine/threonine kinases and underexpression of enzymes of the MAPK pathway. Acquisition of a selective <em>growth</em> advantage after viral integration might be explained by a major shift from a MAPK pathway to cell-cycle dysregulation (G(<em>2</em>)/M).

Sunburn cells (SBCs) appear in the epidermis shortly after acute UV damage, especially after exposure to UVB light. As yet, the mode of their formation remains to be satis<em>factor</em>ily elucidated. In order to characterize these cells, the expression of various markers of epidermal differentiation following UV exposure was investigated using immunhistochemical procedures. These were applied to paraffin-embedded (microwave technique) and frozen specimens of human skin <em>2</em>4 h after irradiation with 4 times the minimal erythema doses(MED). Normal nonirradiated skin without irradiation served as the control. We used a battery of antibodies directed against the following: cytokeratins (CKs) 5, 10, 17, and 19, actin, cell-adhesion proteins (desmoplakins, desmogleins), markers of terminal epidermal differentiation (filaggrin, involucrin and loricrin), markers of proliferation (PCNA, MIB, K6,16), a marker of endocytosis (clathrin) and markers of cell <em>growth</em>, (transforming <em>growth</em> <em>factor</em> [TGF-alpha]) and B-cell leukemia/lymphoma-<em>2</em> [bcl-<em>2</em>]. After UV irradiation it was found that CK 5, which is typically confined to basal <em>keratinocytes</em>, was also expressed in suprabasal <em>keratinocytes</em>. The CKs 1 and 10/11 exhibit a normal suprabasal localization, but suprisingly, SBCs were negative for these CKs. Although CK 6,16, and 17 are not usually found in normal epidermis, UVB exposure induced their expression in suprabasal <em>keratinocytes</em>, but again failed to elicit their expression in SBCs. Antibodies specific for markers of late epidermal differentiation (filaggrin, involucrin and loricrin), cell-junction proteins (desmogleins, desmoplakins), proliferation (PCNA and MIB), and endocytosis (clathrin) also failed to produce positive staining of SBCs. Even though TGF-alpha immunoreactivity became detectable in most <em>keratinocytes</em> after UV exposure, this was not the case for SBCs. The number of basally located dendritic cells, most probably melanocytes, exhibiting bcl-<em>2</em> staining was markedly reduced 6 and 1<em>2</em> h after irradiation as compared with normal skin. SBCs do not express any late differentiation markers, but they do contain proteins typical of basal <em>keratinocytes</em> (CK 5). It can be concluded that SBCs do not develop beyond a more basal-like differentiation pattern, probably as a result of cell death and migration through the epidermis.

The insulin-like <em>growth</em> <em>factor</em> (IGF) system plays an important role in a number of disease states, such as cancer and psoriasis, through its ability to modulate cell proliferation, attachment, and migration. The type-1 IGF and type-<em>2</em> IGF receptors, as well as six IGF-binding proteins (IGFBP-1-6), have well-established roles in mediating IGF activity. Additionally, it's been demonstrated that IGF-II binds directly to the extracellular matrix protein vitronectin (VN), whereas IGF-I does not. IGFBP-5, however, has been recently demonstrated to facilitate the binding of IGF-I to VN. The aim of this study was to determine whether the interaction between IGF, IGFBP, and VN modulates human <em>keratinocyte</em> function. Functional assays demonstrated that both the IGF-II:VN and IGF-I:IGFBP-5:VN complexes resulted in significantly enhanced protein synthesis and cell migration through 1<em>2</em> microm pore Transwells in skin <em>keratinocytes</em> (HaCAT). Furthermore, the IGF-II:VN complex significantly enhanced human corneal epithelial (HCE) cell protein synthesis. Interestingly, the IGF-II:VN complex did not effect either HCE cell migration or attachment. This is the first study to demonstrate a functional role for the interaction between IGF and VN in human <em>keratinocytes</em>. Moreover, these results suggest that IGF-II:VN and IGF-I:IGFBP-5:VN complexes may be useful in situations where enhanced <em>keratinocyte</em> cell migration and proliferation is required, such as in wound healing and skin regeneration.

Normal human <em>keratinocytes</em> synthesize and release nerve <em>growth</em> <em>factor</em> (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing <em>keratinocyte</em> apoptosis. We report here that apoptosis is induced in normal human <em>keratinocytes</em> in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K<em>2</em>5<em>2</em>, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated <em>keratinocytes</em>, the apoptotic process starts at 96 h, and is maximal at 1<em>2</em>0 h. After K<em>2</em>5<em>2</em> treatment, apoptosis starts at 48 h and peaks at 1<em>2</em>0 h. Because the product of the bcl-<em>2</em> proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic <em>keratinocytes</em>. We found that both K<em>2</em>5<em>2</em> and anti-NGF antibody strikingly downregulate bcl-<em>2</em> expression, starting at 7<em>2</em> h. Furthermore, HaCat <em>keratinocytes</em> stably transfected with a plasmid containing bcl-<em>2</em> cDNA fail to undergo apoptosis when treated with K<em>2</em>5<em>2</em>. These findings show that autocrine NGF acts as a survival <em>factor</em> for human <em>keratinocytes</em> in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-<em>2</em>.

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve <em>growth</em> <em>factor</em> (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P<em>2</em>X(5), P<em>2</em>X(7), P<em>2</em>Y(1) and P<em>2</em>Y(<em>2</em>) receptors, and immunostaining of <em>keratinocytes</em> was quantified using optical densitometry. In normal rat epidermis, P<em>2</em>Y(1) and P<em>2</em>Y(<em>2</em>) receptors were found in the basal layer where <em>keratinocytes</em> proliferate; P<em>2</em>X(5) receptors were associated with proliferating and differentiating epidermal <em>keratinocytes</em> in basal and suprabasal layers; P<em>2</em>X(7) receptors were associated with terminally differentiated <em>keratinocytes</em> in the stratum corneum. In the regenerating epidermis of denervated wounds, P<em>2</em>Y(1) receptor protein expression was significantly increased in <em>keratinocytes</em> (P<0.001) but P<em>2</em>Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in <em>keratinocytes</em>. P<em>2</em>X(5) (P>0.001) and P<em>2</em>Y(1) receptor protein (P<0.001) expression in <em>keratinocytes</em>. P<em>2</em>X(7) receptors were absent in all experimental wound healing preparations. P<em>2</em>X(5), P<em>2</em>X(7), P<em>2</em>Y(1) and P<em>2</em>Y(<em>2</em>) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.

We tested the hypothesis that foreign particles shed from filters can accelerate the rate of protein aggregation and particle formation during agitation stress. Various types and brands of syringe filters were tested. Particle counts and size distribution (≥1 µm) in buffer alone or in solutions of <em>keratinocyte</em> <em>growth</em> <em>factor</em> <em>2</em> (KGF-<em>2</em>) were determined with a micro-flow imaging. Submicron particle populations were characterized by dynamic light scattering. Loss of soluble protein during filtration or postfiltration incubation was determined by ultraviolet spectroscopy and bicinchoninic acid protein assay. There was a wide range (from essentially none to >100,000/mL) in the counts for at least 1 µm particles shed into buffer or KGF-<em>2</em> solution from the different syringe filters (with or without borosilicate glass microfibers). Filtration of KGF-<em>2</em> with units containing glass microfibers above the membrane resulted in <em>2</em>0%-80% loss of protein due to adsorption to filter components. Filtration with systems containing a membrane alone resulted in 0%-<em>2</em>0% loss of KGF-<em>2</em>. Effects of <em>2</em>4-h postfiltration incubation were tested on KGF-<em>2</em> solution filtered with polyether sulfone membrane filters. Loss of soluble protein and formation of particles during agitation were much greater than that in control, unfiltered KGF-<em>2</em> solutions. Similar acceleration of protein aggregation and particle formation was observed when unfiltered KGF-<em>2</em> solution was mixed with filtered buffer and agitated. Particle shedding from syringe filters--and the resulting acceleration of protein aggregation during agitation--varied greatly among the different syringe filters and individual units of a given filter type. Our results demonstrate that nanoparticles and microparticles shed from the filters can accelerate protein aggregation and particle formation, especially during agitation.

Healing of corneal wounds is a complex process involving epithelial, keratocyte, and endothelial interactions that are affected by their associations with wound bed matrix and by cytokine availability and activation. The spectrum of possible cellular-matrix-<em>growth</em> <em>factor</em> interactions is indeed great and <em>growing</em>. Several of the significant contributions made during the past year include development of an organotypic organ culture model of the cornea that allows in vitro assembly of the epithelial extracellular matrix-anchoring complex, demonstration of epithelial synthesis of Bowman's layer collagens, demonstration of transforming <em>growth</em> <em>factor</em>-beta <em>2</em>'s inhibition of stromal cell collagenase synthesis, and demonstration of the paracrine pathway of <em>keratinocyte</em> <em>growth</em> <em>factor</em> action in the cornea.

Investigations of the pathways regulating normal <em>growth</em> of epithelial cells have revealed the existence of two major <em>growth</em>-<em>factor</em> signaling cascades required for proliferation. One pathway is activated by IGF-1 or high insulin concentration. The other is triggered by EGF, TGF alpha, or members of the FGF family, including the recently discovered epithelial-cell-specific <em>growth</em> <em>factor</em>, designated <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). Its expression pattern in vivo suggests that KGF plays an important normal physiologic role as a stromal effector of epithelial cell proliferation. Oncogenes, which represent constitutively activated forms of genes critically involved in <em>growth</em>-<em>factor</em> signaling pathways, specifically abrogate the requirement for mitogens of the EGF pathway. Examples of such genes include the erbB/EGF receptor and erbB-<em>2</em>, which encode structurally related receptor proteins and are often amplified and/or overexpressed in epithelial malignancies. Employing reduced stringency hybridization with v-erbB as a probe, we recently identified a third member of this receptor family, designated erbB-3. cDNA cloning revealed a predicted 148-kD transmembrane polypeptide with structural features similar to those of the EGF receptor. Normal erbB-3 expression in <em>keratinocytes</em> and glandular epithelium suggests its physiologic role in these cell types. Moreover, markedly elevated erbB-3 mRNA levels in certain mammary tumor cell lines suggest that increased erbB-3 expression may also play a role in some human epithelial malignancies.

BACKGROUND

Keratinocyte growth factor (KGF) increases intestinal growth and is expressed by intestinal intraepithelial lymphocytes (IEL). Because total parenteral nutrition (TPN) leads to villus atrophy and a loss of epithelial function, we hypothesized that KGF administration could reverse these changes.

METHODS

Mice were randomized into three groups: oral feeding (Control); TPN; or TPN with recombinant human KGF. Mice were killed at 7 days, and the small bowel was harvested for histology, DNA, and protein content analysis. Epithelial cell proliferation was studied by 5-bromo-2-deoxyuridine (BrdU) incorporation, and apoptosis was detected by flow cytometry with Annexin V staining. Epithelial ion transport function was studied by Ussing chambers. Epithelial barrier function was assessed with transepithelial resistance and transmural passage of 3H-mannitol. Epithelial KGF receptors expression was studied by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.

RESULTS

TPN decreased intestinal DNA, protein content, villus height, and crypt cell proliferation. TPN also resulted in an increase in epithelial cell apoptosis. KGF administration significantly stimulated the recovery of mucosal structures including intestinal protein and DNA content, villus height, and crypt cell proliferation, and decreased epithelial apoptosis. KGF also up-regulate the epithelial KGF receptor expression. Moreover, KGF attenuated the TPN-induced increase in ion transport and increased the epithelial barrier function.

CONCLUSIONS

KGF administration reversed many of the adverse epithelial changes associated with TPN administration. Additionally, KGF up-regulated epithelial KGF receptor expression. It is possible that KGF may have a therapeutic efficacy in patients who are receiving TPN.

OBJECTIVE

Low level laser irradiation (LLLI) is used to promote wound healing. Molecularly it is known to stimulate mitochondrial membrane potential (MMP), cytokine secretion, and cell proliferation. This study was designed to determine the influence of LLLI on the kinetics of MMP stimulation and decay, specific cytokine gene expression, and subcellular localization of promyelocytic leukemia (PML) protein on HaCaT human keratinocytes.

METHODS

The cells were irradiated by a 780 nm titanium-sapphire (Ti-Sa) laser with 2 J/cm(2) energy density. MMP was monitored with Mitotracker, a mitochondrial voltage-sensitive fluorescent dye. Cytokine gene expression was carried out using semi-quantitative-reverse transcription polymerase chain reaction. Subcellular localization of PML protein, a cell-cycle checkpoint protein, was determined using immunofluorescent staining.

RESULTS

The fluorescence intensity of MMP was increased immediately after the end of LLLI by 148 +/- 6% over control (P<0.001). Subsequently it decayed, reaching 51 +/- 14% of the control level (P < 0.01) within 200 minutes. This decay was characterized by an exponential curve (R = 0.96) with a lifetime of 79 +/- 36 minutes (P < 0.05). Following irradiation, the expression of interleukin-1alpha, interleukin-6, and keratinocyte growth factor (KGF) genes were transiently upregulated; but the expression of the proinflammatory gene interleukin-1beta, was suppressed. The subnuclear distribution of PML was altered from discrete domains to its dispersed form within less than 1 hour after LLLI.

CONCLUSIONS

These changes reflect a biostimulative boost that causes a shift of the cell from a quiescent to an activated stage in the cell cycle heralding proliferation and suppression of inflammation. Further characterization of MMP kinetics may provide a quantitative basis for assessment of the effect of LLLI in the clinical setting.

Cholesterol has been recently suggested to regulate the early steps of <em>keratinocyte</em> differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal <em>growth</em> <em>factor</em> receptor (EGFR), human epidermal <em>growth</em> <em>factor</em> receptor <em>2</em> (HER<em>2</em>), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured <em>keratinocytes</em>, using a treatment with methyl-beta-cyclodextrin (MbetaCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MbetaCD treatment induces phosphorylation of EGFR, HER<em>2</em>, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MbetaCD-induced phosphorylation of EGFR, HER<em>2</em>, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during <em>keratinocyte</em> differentiation. We found that MbetaCD treatment induces a prolonged phosphorylation of p38 in general and p38alpha in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MbetaCD, but not by a p38delta-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38alpha/beta.

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering <em>growth</em> <em>factors</em> to promote skin wound healing. The approaches involving transient delivery of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (<em>2</em>) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver <em>growth</em> <em>factors</em> to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

Treatment of human HaCaT <em>keratinocytes</em> with corticotropin-releasing hormone modulates cell proliferation and expression of inflammation markers. In this study we report that corticotropin-releasing hormone also inhibits nuclear <em>factor</em>-kappaB binding and transcriptional activity. Incubating cells in the absence of <em>growth</em> <em>factors</em> increased nuclear <em>factor</em>-kappaB activity; this effect was significantly attenuated by corticotropin-releasing hormone. Specifically, corticotropin-releasing hormone downregulated p50/p50 and p50/p65 dimers of nuclear <em>factor</em>-kappaB, diminished kappaB-driven CAT reporter gene activity and inhibited IkappaB-beta degradation. Moreover, corticotropin-releasing hormone inhibited the trans-cription of the nuclear <em>factor</em>-kappaB responsive genes, interleukin-<em>2</em> and heat shock protein 90.

Psoriasis is an inflammatory skin disorder with hyperproliferation of <em>keratinocytes</em>, that can be the result of insufficient inhibitory effect of transforming <em>growth</em> <em>factors</em>-beta (TGF-beta). The aim of this study was to evaluate an association between TGF-beta(1) and -beta(<em>2</em>) in plasma or scales from psoriatic lesions and the severity of the disease. TGF-beta concentrations were measured with an enzyme immunoassay in 41 patients with psoriasis. The mean plasma concentrations of TGF-beta(1) and TGF-beta(<em>2</em>) in patients were: 15.7 +/- 1.4 and 0.15 +/- 0.0<em>2</em> ng/ml respectively. It was also detectable in scales and varied from <em>2</em>4 to 1159 and from 0 to <em>2</em>.95 pg/mg protein respectively. Plasma TGF-beta(1) correlated significantly with psoriasis area and severity index (PASI). Significant correlation was also demonstrated between TGF-beta(1) concentration in scales and sedimentation rate or the disease duration. There were no correlation between PASI and plasma TGF-beta(<em>2</em>), scales TGF-beta(1) and TGF-beta(<em>2</em>). The highest mean concentration of TGF-beta(1) in scales of patients with mild form of the disease (<em>2</em>03 +/- 65 pg/mg protein) and the lowest in severe form (147 +/- 54 pg/mg protein) have been shown. These findings demonstrated association between PASI and plasma levels of TGF-beta(1), that should be considered as a possible indicator of psoriasis activity.

Recently, glucagon-like peptide <em>2</em> has emerged as a potent stimulator of epithelial <em>growth</em>, joining insulin-like <em>growth</em> <em>factor</em> I, hepatocyte <em>growth</em> <em>factor</em> and <em>keratinocyte</em> <em>growth</em> <em>factor</em> as potential treatment modalities for intestinal disorders associated with loss of mucosal mass, such as short bowel syndrome. Investigations into other members of the expanded epidermal <em>growth</em> <em>factor</em> peptide family, the development of more potent peptide analogues, and advances in the development of enterally administered bioactive <em>growth</em> <em>factor</em> formulations further expands the repertoire of epithelial <em>growth</em> <em>factors</em> applicable to conditions associated with epithelial insufficiency.

During fetal and neonatal development and experimental obstruction, the bladder wall undergoes changes in both the amount and composition of the urothelium, extracellular matrix, and smooth muscle. We hypothesize that cell-cell signaling among the different layers of the bladder wall mediates these changes. <em>Growth</em> <em>factors</em> likely to be involved in this process are <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and transforming <em>growth</em> <em>factor</em> (TGF)-alpha, -beta <em>2</em>, and -beta 3. Whole rodent bladders were analyzed by RNase protection assays for KGF, KGF receptor, TGF alpha, epidermal <em>growth</em> <em>factor</em> receptor, and TGF beta <em>2</em> and -beta 3 transcripts at Fetal Day 14 (before smooth muscle differentiation) and Fetal Day 18 (after smooth muscle differentiation), at birth, and 60 days postnatal. <em>Growth</em> <em>factor</em> transcripts were also analyzed in partially obstructed rodent bladders and in sham-operated animals. TGF beta <em>2</em> and -beta 3 mRNA expression decreased as a function of gestational age, whereas TGF alpha mRNA increased. KGF mRNA was low before smooth muscle differentiation at 14 days' gestation, then increased. The mRNA of receptors for KGF and EGF remained essentially unchanged throughout bladder development. In bladders subjected to partial urethral outlet obstruction, there was a <em>2</em>-fold increase in mRNA for TGF beta <em>2</em>, a 5-fold increase in TGF beta 3, and a 10-fold increase TGF alpha mRNA. In contrast, there was no change in transcripts for either KGF or receptors for KGF and epidermal <em>growth</em> <em>factor</em>. Immunohistochemical localization of the protein for these <em>growth</em> <em>factors</em> showed selective localization to the epithelium and/or smooth muscle for TGF beta <em>2</em> and -beta 3, whereas TGF alpha and the epidermal <em>growth</em> <em>factor</em> receptor localized throughout the bladder wall. In conclusion, <em>growth</em> <em>factor</em> mRNA expression is modulated in bladder development and obstruction, which implies a possible mechanistic role of <em>growth</em> <em>factors</em> for the observed changes in the bladder wall and extracellular matrix.

Capsiate, one of the major capsaicinoids, is nonpungent and present in sweet pepper. We investigated the effects of capsiate on the ultraviolet B (UVB)-induced inflammatory response in skin and its molecular mechanisms. Capsiate-pretreated human <em>keratinocytes</em> inhibited intracellular reactive oxygen species (ROS), which activate the mitogen-activated protein kinase and nuclear <em>factor</em>-kappaB (NF-kappaB) pathways. Therefore, we determined the effects of capsiate on these pathways. Capsiate inhibited UVB-induced cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression, extracellular signal-related kinase 1/<em>2</em> phosphorylation, nuclear translocation of NF-kappaB, and the expression of proinflammatory cytokines and potent angiogenic <em>factors</em>, including vascular endothelial cell <em>growth</em> <em>factor</em> and matrix metalloproteinase-<em>2</em> (MMP-<em>2</em>) and MMP-9. In addition, capsiate inhibited UVB-induced epidermal <em>growth</em> <em>factor</em> receptor (EGFR) activation, which reduces the levels of proinflammatory cytokines and angiogenic <em>factors</em>. We also investigated the photoprotective effects of capsiate in vivo. Topical treatment with capsiate significantly decreased UVB-induced skin damage and inhibited the expression of COX-<em>2</em>, proinflammatory cytokines, and angiogenic <em>factors</em>, including platelet/endothelial cell adhesion molecule-1 and intercellular adhesion molecule-1. Inhibition of Src kinase activity and ROS may inhibit the EGFR activation. Therefore, capsiate may protect the skin from UVB-induced adverse effects and these results provide a molecular basis for understanding its effects on inflammation and angiogenesis.

The results of a thymidine incorporation assay were compared with direct measurement of cell number in assessment of proliferative <em>growth</em> of human <em>keratinocytes</em> in monolayer culture. <em>Keratinocytes</em> were cultured in supplemented MCDB 153 medium in 0.1 mM Ca<em>2</em>+, and plated in <em>2</em>4-well trays. The ability of insulin, placental extract, and epidermal <em>growth</em> <em>factor</em> to enhance <em>growth</em> and thymidine incorporation were compared. Autoradiography was performed to determine the percentage of cells with labeled nuclei. Epidermal <em>growth</em> <em>factor</em> increased thymidine incorporation under the conditions of the assay, and placental extract increased incorporation by up to 50-fold, since the control cells plated in the absence of epidermal <em>growth</em> <em>factor</em> and other <em>growth</em> <em>factors</em> survived but proliferated minimally. Both cell number and thymidine incorporation showed similar concentration dependence upon insulin and placental extract. If placental extract was added to cells plated <em>2</em>8 h earlier, incorporation was maximal after 17 h in the presence of the extract. If cells were plated in the presence of the extract, 85% of nuclei were shown by autoradiography to be labeled after <em>2</em>3 h, but only <em>2</em>4% of nuclei were labeled in the absence of the extract. A plating density of 10(4) cells/<em>2</em>-cm<em>2</em> well was optimal. The assay permits rapid identification of <em>growth</em>-promoting fractions without prolonged <em>growth</em> periods, and is a valid indicator of these agents in <em>keratinocyte</em> cultures.

Cultured primary human <em>keratinocytes</em> were the first non-cancer-derived cell type reported to produce the humoral hypercalcemia <em>factor</em>, parathyroid hormone-related protein (PTHrP). Emerging evidence suggests that only a subset of <em>keratinocytes</em> produce high levels of PTHrP in vivo. We found that the PTHrP mRNA content of intact human skin was minimal, whereas transcripts were easily detectable in primary <em>keratinocytes</em> derived from those skin samples. We hypothesized that conditions associated with <em>growth</em> in culture activated PTHrP gene expression in primary <em>keratinocytes</em>. In culture, <em>keratinocytes</em> produce a number of epidermal <em>growth</em> <em>factor</em> (EGF)-like ligands (transforming <em>growth</em> <em>factor</em>-alpha, heparin binding-EGF and amphiregulin) and their receptor, ErbB1. Treatment of <em>keratinocytes</em> with a specific erbB1 inhibitor (PD153035) reduced PTHrP mRNA levels by >80% in rapidly <em>growing</em> <em>keratinocytes</em>. Treatment of <em>keratinocytes</em> with reagents that neutralize amphiregulin reduced PTHrP mRNA levels by approximately 60%. Blockade of erbB1 signaling reduces transcription from the endogenous PTHrP P3-TATA promoter. The Ets transcription <em>factor</em>-binding site, 40 bases upstream of the P3 promoter, is required for baseline expression of PTHrP reporter gene constructs in <em>keratinocytes</em>; in addition, cotransfection of Ets-1 and Ets-<em>2</em> expression vectors activate the reporter gene constructs. Finally, disruption of both ras and raf signaling reduce reporter gene expression by 80%, suggesting that ErbB1 signaling is mediated by the classic ras/MAP kinase pathway. These findings suggest that acquisition of EGF-like ligand expression has the potential to substantially activate PTHrP gene expression in the epidermis.

The effects of human recombinant transforming <em>growth</em> <em>factor</em> (TGF)-beta 1 were determined on PTH-related protein (PTHrP) production and messenger RNA (mRNA) expression by a canine squamous carcinoma cell line (SCC <em>2</em>/88) in vitro. TGF-beta increased PTHrP production in a dose- and time-dependent manner (P < 0.05) as measured by RIA, and the effects of TGF-beta treatment persisted up to 7<em>2</em> h after removal. TGF-beta increased PTHrP production by SCC <em>2</em>/88 cells until cellular confluence, at which time there was no longer a significant increase compared to control. Actinomycin D inhibited the TGF-beta-mediated increase in PTHrP production, suggesting that TGF-beta acted in part by increasing gene transcription. SCC <em>2</em>/88 cells also produced active TGF-beta as measured by a [3H]thymidine incorporation assay in mink lung epithelial cells. Exposure of SCC <em>2</em>/88 cells to a neutralizing anti-TGF-beta monoclonal antibody decreased (up to 50%, P < 0.01) basal PTHrP production. TGF-beta increased PTHrP mRNA expression in a dose- and time-dependent manner as evaluated by northern blot analysis. Postconfluent SCC <em>2</em>/88 cells expressed little mRNA for PTHrP, and there was only a minimal increase in PTHrP mRNA expression in postconfluent TGF-beta-treated cells. These results indicate that TGF-beta increased PTHrP production and mRNA expression in malignant <em>keratinocytes</em> and suggest that TGF-beta may be an important <em>factor</em> in the pathogenesis of humoral hypercalcemia of malignancy.

OBJECTIVE

Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration.

METHODS

Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model.

RESULTS

The optimized glue is based on the fibrinogen component of TISSUCOL, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group).

CONCLUSIONS

COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.

Matrilysin-<em>2</em> (matrix metalloproteinase (MMP)-<em>2</em>6) is a small protein of the MMP family expressed in some epithelial carcinomas and normal tissues. We studied its role in benign skin disorders characterized by epithelial proliferation, in wound repair, skin cancer, and regulation in <em>keratinocyte</em> (KC) cultures. MMP-<em>2</em>6 is expressed by laminin-5-positive KC in the migrating area during wound repair, in benign skin disorders characterized by inflammation and microdisruptions of basement membrane, but in intact skin only in hair follicles. It was detected in occasional atypical KC in pre-malignant lesions but not in basal cell cancer islands. Although MMP-<em>2</em>6 was expressed in grades I and II squamous cell cancers (SCC), it was not present in dedifferentiated grade III tumors. MMP-<em>2</em>6 was neither co-expressed with its close homologue matrilysin-1 nor with the proliferation marker Ki-67. But in tissue samples it either co-localized or was detected in adjacent cells of same regions with the tumor suppressor p16. In KC and HaCaT cell cultures, 1<em>2</em>-phorbol-13-myristate-acetate, epidermal <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em>-alpha, transforming <em>growth</em> <em>factor</em>-beta1, interleukin-1 (IL-1)beta, IL-6, insulin-like <em>growth</em> <em>factor</em>, gamma-IFN, retinoic acid, dexamethasone, four matrices or ras-transformation were unable to upregulate MMP-<em>2</em>6 expression. The expression pattern of MMP-<em>2</em>6 suggests that it may be upregulated in basal KC even without tumorigenesis because of altered cell-matrix interactions and inflammation and, unlike most MMP, becomes downregulated during histological dedifferentiation of SCC. Thus, lack of MMP-<em>2</em>6 in SCC could be a marker of aggressive <em>growth</em>.

The purpose of this study was to determine the efficacy of a novel human protein, <em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), in a model of murine colitis induced by ad libitum exposure to a 4% solution of dextran sulfate sodium (DSS) in the drinking water. Initial evaluation of KGF-<em>2</em> was based on its ability to reduce weight loss, stool score, and histological score in mice exposed to DSS for 7 days. When KGF-<em>2</em> (0.1-10.0 mg/kg i.p. or s.c.) was injected daily into DSS-treated mice from day 0 to 7, it significantly reduced all three parameters in a dose-response fashion, with a minimum effective dose of between 1 and 3 mg/kg. When KGF-<em>2</em> was given therapeutically, starting 4 days after initiation of the 7-day DSS treatment, the 3- but not the 0.5-mg/kg dose significantly enhanced weight recovery after discontinuation of DSS treatment. When DSS treatment was prolonged beyond the normal 7 days, therapeutic intervention on day <em>2</em> or 4 also significantly reduced mortality, weight loss, and stool score at the 1- and 3-mg/kg dose. Therapeutic treatment also resulted in reduction of colon myloperoxidase levels by more than 50%. These experiments suggest that KGF-<em>2</em> may be clinically useful in the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

Human involucrin (hINV), first appears in the cytosol of <em>keratinocytes</em> and ultimately cross-linked to membrane proteins via transglutaminase and forms a protective barrier as an insoluble envelope beneath the plasma membrane. Although the function and evolution of involucrin is known, the regulation of its gene expression is not well understood. An analysis of the hINV gene sequence, upstream of the transcription start site (-534 to +1 nt) revealed the presence of potential sites for binding of lens epithelium-derived <em>growth</em> <em>factor</em> (LEDGF); stress response element (STRE; A/TGGGGA/T) and heat shock element (HSE; nGAAn). We reported earlier that LEDGF activates stress-associated genes by binding to these elements and elevates cellular resistance to various stresses. Here, gel-shift and super-shift assays confirm the binding of LEDGF to the DNA fragments containing HSEs and STREs that are present in the involucrin gene promoter. Furthermore, hINV promoter linked to CAT reporter gene, cotransfected in human corneal simian virus 40-transformed <em>keratinocytes</em> (HCK), was transactivated by LEDGF significantly. In contrast, the activity of hINV promoter bearing mutations at the WT1 (containing HSE and STRE), WT<em>2</em> (containing STRE) and WT3 (containing STRE) binding sites was diminished. In addition, in HCK cell over-expressing LEDGF, the levels of hINV mRNA and hINV protein are increased by four to five-fold. LEDGF is inducible to oxidants. Cells treated with 1<em>2</em>-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate production of H(<em>2</em>)O(<em>2</em>), showed higher levels of LEDGF mRNA. Furthermore, our immunohistochemical studies revealed that hINV protein is found in the cytoplasm of HCK cells over-expressing LEDGF, but not detectable in the normal HCK cells or HCK cells transfected with vector. This regulation appears to be physiologically important, as over-expression of HCK with LEDGF increases the expression of the endogenous hINV gene and may provide new insight to understand the molecular mechanism of transcriptional regulation of this gene. LEDGF may play an important role in establishing an important barrier in corneal <em>keratinocytes</em> by maintaining epidermal turn-over rate, and protecting HCKs against stress.