BACKGROUND

Proteoglycans (PGs) represent a large family of complex molecules. They are found either as integral membrane components or constituents of the extracellular matrix. Their protein backbones are linked to different glycosaminoglycans, such as dermatan-, chondroitin-, keratan- or heparan sulphate. The molecules have specific functions during developmental processes as well as in diseases, such as cancer and inflammation.

OBJECTIVE

The expression patterns of various cell-associated heparan and chondroitin/dermatan-sulphate PGs in human skin and chronic venous ulcers were investigated.

METHODS

Tissue sections from 11 patients with chronic venous ulcers were used in this study. Monoclonal antibodies were used for detection of the proteoglycans syndecan-1, -<em>2</em> and -4, glypican, CD44 and perlecan.

RESULTS

The different PGs exhibited individual staining patterns. Syndecan-1 and -4 and glypican expression in chronic ulcers differed from the staining in normal skin. Whereas the expression of syndecan-4 and glypican in intact skin was mostly in the pericellular regions of keratinocytes, the epidermal cells from the wound edge contained mostly intracellular PGs. In the wound edge, syndecan-4 was predominantly expressed by epidermal basal layer cells. Syndecan-1 was less expressed at the epidermal wound margins. PGs bind growth factors, regulate proteolytic activity and act as matrix receptors.

CONCLUSIONS

The altered expression patterns of glypican and syndecan-1 and -4 in chronic ulcers reflect their possible roles during inflammation and cell proliferation. Hence, analysis of PG expression should be of interest in future studies on normal as well as defective wound healing.

High-risk human papillomaviruses (hr-HPV) establish persistent infections in <em>keratinocytes</em>, which can lead to cancer of the anogenital tract. Interferons (IFNs) are a family of secreted cytokines that induce IFN-stimulated genes (ISGs), many of which display antiviral activities. Transcriptome studies have indicated that established hr-HPV-positive cell lines display a reduced expression of ISGs, which correlates with decreased levels of interferon kappa (IFN-κ), a type I IFN constitutively expressed in <em>keratinocytes</em>. Prior studies have also suggested that IFN-β has anti-hr-HPV activity but the underlying mechanisms are not well understood. The downregulation of IFN-κ by hr-HPV raises the possibility that IFN-κ has anti-HPV activity. Using doxycycline-inducible IFN-κ expression in CIN61<em>2</em>-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrated that IFN-κ inhibits the <em>growth</em> of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited at 4 to 6 h after IFN-κ expression. This was also observed with recombinant IFN-β, suggesting a common mechanism of IFNs. Transcriptome sequencing (RNA-seq) analysis identified 1,367 IFN-κ-regulated genes, of which <em>2</em><em>2</em>1 were modulated>><em>2</em>-fold. The majority of those (71%) matched known ISGs, confirming that IFN-κ acts as a bona fide type I IFN in hr-HPV-positive <em>keratinocytes</em>. RNA interference (RNAi) and cotransfection experiments indicated that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-κ. Consistent with published data showing that Sp100 acts as a restriction <em>factor</em> for HPV18 infection, our results suggest that hr-HPV target IFN-κ to prevent Sp100 expression and identify Sp100 as an ISG with anti-HPV activity.

OBJECTIVE

High-risk HPV can establish persistent infections which may progress to anogenital cancers. hr-HPV interfere with the expression of interferon (IFN)-stimulated genes (ISGs), which is due to reduced levels of IFN-κ, an IFN that is constitutively expressed in human keratinocytes. This study reveals that IFN-κ rapidly inhibits HPV transcription and that this is due to the induction of Sp100 proteins. Thus, Sp100 represents an ISG for hr-HPV.

During bladder development, undifferentiated mesenchymal and epithelial cells undergo an orderly sequence of differentiation defined by the expression of smooth-muscle (alpha-actin, myosin, vinculin, desmin, vimentin, and laminin) and epithelial (cytokeratins 5, 7, 8, 14, 18 and 19) protein markers. This process requires mesenchymal-epithelial interactions with bladder epithelium (urothelium) necessary for the differentiation of bladder smooth muscle. Peptide <em>growth</em> <em>factors</em> such as <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and transforming <em>growth</em> <em>factors</em> (TGF) alpha and beta are likely candidates as mediators of these mesenchymal-epithelial interactions. Transcripts for KGF, TGF alpha, and TGF beta are regulated during bladder development and during smooth-muscle hypertrophy secondary to bladder-outlet obstruction. Finally, two experimental bladder models--(1) partial outlet obstruction and (<em>2</em>) regeneration of bladder smooth muscle into an acellular tissue matrix--are described in the context of mesenchymal-epithelial interactions in the bladder.

Trefoil <em>factor</em> family 3 (TFF3) is secreted in saliva. The peptide improves the mechanical and chemical resistance of mucins, and it may act as a motility signal for oral <em>keratinocytes</em> during wound healing. This study aimed to identify novel functions of TFF3 in oral <em>keratinocytes</em>. To achieve this, we used phosphoprotein and messenger RNA (mRNA) arrays to compare TFF3-treated and untreated oral <em>keratinocytes</em>. Analysis of the phosphoprotein array indicated that TFF3 signals through the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK1/<em>2</em>), and through the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway. Microarray analysis of mRNA showed that TFF3 stimulation induced changes in the expression of genes functionally related to cell death/survival, cell <em>growth</em> and proliferation, and cell movement. The reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the transcription of some immediate-early genes (IEGs) was downregulated, whereas the IEGs FBJ osteosarkoma oncogene (FOS) and C-MYC binding protein (MYCBP<em>2</em>) were transiently upregulated by TFF3 stimulation. Together, the results of the arrays indicate that TFF3 is a modifying <em>factor</em> in pathways regulating cell survival, cell <em>growth</em> and proliferation, and cell migration of oral <em>keratinocytes</em>. Trefoil <em>factor</em> family 3 may therefore promote oral wound healing and it should be considered for the treatment of oral ulcerating diseases, or of other diseases.

Regulation of cell <em>growth</em> and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on <em>growth</em> suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal <em>growth</em> <em>factor</em>-triggered <em>growth</em> signal pathway with cytosolic phospholipase A(<em>2</em>) (cPLA(<em>2</em>)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human <em>keratinocytes</em> (NHK). Ca(<em>2</em>+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(<em>2</em>), resulting in inhibition of cPLA(<em>2</em>) activity, which is essential for the <em>growth</em> of NHK. On exposure of NHK to epidermal <em>growth</em> <em>factor</em>, ANXA1 was cleaved solely at Trp(1<em>2</em>), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell <em>growth</em> disorders.

Disruption of the murine permeability barrier by solvents or tape stripping stimulates a homeostatic repair response that includes increased epidermal DNA synthesis. To identify potential mediators of the increase in DNA synthesis, we have measured epidermal levels of mRNAs encoding various <em>growth</em> <em>factors</em> after acute barrier disruption. In this study, mRNAs for amphiregulin and nerve <em>growth</em> <em>factor</em> were each shown to increase over controls at 30 min, reach peak levels of 1<em>2</em>- to 30-fold at 1-<em>2</em> h, and return to control levels by 6 h after tape stripping. A similar time course for the increase of amphiregulin and nerve <em>growth</em> <em>factor</em> mRNAs was observed after an unrelated form of barrier disruption, i.e., acetone treatment. Furthermore, artificial restoration of the barrier by Latex occlusion, immediately following barrier disruption by acetone treatment, inhibited the increase in epidermal amphiregulin and nerve <em>growth</em> <em>factor</em> mRNA levels, indicating that barrier status regulates the production of these <em>growth</em> <em>factors</em>. In contrast, mRNA levels of transforming <em>growth</em> <em>factor</em>-beta1, an inhibitory <em>growth</em> <em>factor</em>, were unchanged at early times and decreased by 53% (p < 0.0<em>2</em>) 6 h after tape stripping, whereas mRNA levels of transforming <em>growth</em> <em>factor</em>-alpha remained unchanged at all times after acute barrier disruption. These results suggest that barrier disruption stimulates the expression of amphiregulin and nerve <em>growth</em> <em>factor</em>. Together, these regulators of <em>keratinocyte</em> <em>growth</em> and differentiation may be responsible for the increased proliferative response that is associated with barrier disruption.

OBJECTIVE

The presence of regional metastases in head and neck squamous cell carcinoma (HNSCC) patients is a common and adverse event associated with poor prognosis. Understanding the molecular mechanisms that mediate HNSCC metastasis may enable identification of novel therapeutic targets. E-cadherin plays a key role in epithelial intercellular adhesion; its downregulation is a hallmark of the epithelial-to-mesenchymal transition (EMT) (an essential process during tumor progression); and it is associated with invasion, metastasis, and decreased survival. Inflammatory cytokines have been implicated in the progression of HNSCC. Herein, the mechanisms by which the inflammatory mediator, Interleukin-1β (IL-1β), might contribute to EMT in HNSCC is investigated. The pathways involved in E-cadherin regulation in HNSCC had not previously been defined. It is hypothesized that 1) inflammatory mediators upregulate cyclooxygenase-<em>2</em>/prostaglandin E<em>2</em> (COX-<em>2</em>/PGE<em>2</em>), which then in turn regulate E-cadherin expression in HNSCC; and <em>2</em>) PGE<em>2</em> downregulates E-cadherin via transcriptional repressors of E-cadherin (such as Snail) in HNSCC. The outcome of the proposed research will allow us to define how resistance to epidermal <em>growth</em> <em>factor</em> receptor (EGFR)-selective tyrosine kinase inhibitors is mediated and whether the benefits of combination therapy are due to the capacity of COX-<em>2</em> inhibitors to increase E-cadherin expression and thus create a more sensitive target for EGFR TK inhibition.

METHODS

Basic science, molecular biology, animal model, immunohistochemistry.

METHODS

We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a severely compromised immunodeficient (SCID) xenograft model was used to assess the effects in vivo.

RESULTS

COX-<em>2</em>-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-<em>2</em> and E-cadherin was demonstrated in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β caused the downregulation of E-cadherin expression and upregulation of COX-<em>2</em> expression. This effect was blocked in the presence of COX-<em>2</em> small hairpin RNA (shRNA). IL-1β -treated HNSCC cell lines demonstrated a significant decrease in E-cadherin messenger RNA (mRNA) and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. ShRNA-mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. Snail overexpression in normal oral keratinocytes and HNSCC cells is sufficient to drive EMT and confers resistance to erlotinib. In a SCID xenograft model, HNSCC Snail overexpressing cells demonstrated significantly increased primary and metastatic tumor burdens.

CONCLUSIONS

The inflammatory mediator IL-1β modulates Snail and thereby regulates COX-<em>2</em>-dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy.

METHODS

N/A.

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and <em>growth</em> <em>factors</em> and their receptors. The overexpression of MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) is associated with nonhealing wounds, whereas active MMPs-1, -<em>2</em>, -9, and -14 are required for normal wound healing to occur. We describe the synthesis and enzyme inhibition profile of (3R)-3-[([(1S)-<em>2</em>,<em>2</em>-dimethyl-1-(([(1S)-<em>2</em>-methoxy-1-phenylethyl]amino)carbonyl)propyl]amino)carbonyl]-6-(3-methyl-4-phenylphenyl)hexanoic acid (UK-370,106, 7), which is a potent inhibitor of MMP-3 (IC(50) = <em>2</em>3 nM) with >1<em>2</em>00-fold weaker potency vs MMP-1, -<em>2</em>, -9, and -14. MMP-13, which may also contribute to the pathology of chronic wounds, was inhibited about 100-fold less potently by compound 7. Compound 7 potently inhibited cleavage of [(3)H]-fibronectin by MMP-3 (IC(50) = 3<em>2</em>0 nM) but not cleavage of [(3)H]-gelatin by either MMP-<em>2</em> or -9 (up to 100 microM). Compound 7 had little effect, at MMP-3 selective concentrations, on <em>keratinocyte</em> migration over a collagen matrix in vitro, which is a model of the re-epithelialization process. Following iv (rat) or topical administration to dermal wounds (rabbit), compound 7 was cleared rapidly (t(1/<em>2</em>) = <em>2</em>3 min) from plasma, but slowly (t(1/<em>2</em>) approximately 3 days) from dermal tissue. In a model of chronic dermal ulcers, topical administration of compound 7 for 6 days substantially inhibited MMP-3 ex vivo. These data suggest compound 7 is sufficiently potent to inhibit MMP-3-mediated matrix degradation while leaving unaffected cellular migration mediated by MMPs 1, <em>2</em>, and 9. These properties make compound 7 a suitable candidate for progression to clinical trials in human chronic dermal wounds, such as venous ulcers.

BACKGROUND

Wound healing is a complex process involving interactions among a variety of different cell types. The normal wound repair process consists of three phases--inflammation, proliferation, and remodeling that occur in a predictable series of cellular and biochemical events. Wounds are classified according to various criteria: etiology, lasting, morphological characteristics, communications with solid or hollow organs, the degree of contamination. In the last few years many authors use the Color Code Concept, which classifies wounds as red, yellow and black wounds. This paper presents conventional methods of local wound treatment (mechanical cleansing, disinfection with antiseptic solutions, wound debridement--surgical, biological and autolytic; wound closure, topical antibiotic treatment, dressing), as well as general measures (sedation, antitetanous and antibiotic protection, preoperative evaluation and correction of malnutrition, vasoconstriction, hyperglycemia and steroid use, appropriate surgical technique, and postoperative prevention of vasoconstriction through pain relief, warming and adequate volume resuscitation).

UNASSIGNED

<em>Growth</em> <em>factors</em> play a role in cell division, migration, differentiation, protein expression, enzyme production and have a potential ability to heal wounds by stimulating angiogenesis and cellular proliferation, affecting the production and the degradation of the extracellular matrix, and by being chemotactic for inflammatory cells and fibroblasts. There are seven major families of <em>growth</em> <em>factors</em>: epidermal <em>growth</em> <em>factor</em> (EGF), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), insulin-like <em>growth</em> <em>factor</em> (IGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), fibroblast <em>growth</em> <em>factor</em> (FGF), interleukins (ILs), and colony-stimulating <em>factor</em> (CSF). Acute wounds contain many <em>growth</em> <em>factors</em> that play a crucial role in the initial phases of wound healing. The events of early wound healing reflect a finely balanced environment leading to uncomplicated and rapid wound healing. Chronic wounds, for many reasons, have lost this fine balance. Multiple studies have evaluated the effect that exogenously applied <em>growth</em> <em>factors</em> have on the healing of chronic wounds. In the study conducted by Knighton and colleagues, topical application of mixture of various <em>growth</em> <em>factors</em> (PDGF, TGF-beta, PDAF, PF4, PDEGF) demonstrated increased wound healing over controls. Brown and associates demonstrated a decrease in skin graft donor site healing time of 1 day using topically applied EGF. Herndon and ass. used systemic <em>growth</em> hormone in burned children and reduction in healing time made a significant clinical difference by allowing earlier wound coverage and decreasing the duration of hospitalization. The TGF family of <em>growth</em> <em>factors</em> is believed to be primarily responsible for excessive scar formation, especially the beta 1 and beta <em>2</em> isoforms. TGF-beta 3 isoform has recently been described and may have an inhibitory function on scar formation by being a natural antagonist to the TGF-beta 1 and TGF-beta <em>2</em> isoforms. Cytokines, especially interferon-alpha (INF-alpha), INF-alpha, and INF-alpha <em>2</em>b, may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts and reduce the rate of collagen and fibronectin synthesis by reducing the production of mRNA. Expression of nitric oxide synthase (NOS) and heat shock proteins (HSP) have an important role in wound healing, as well as trace elements (zinc, copper, manganese). Applications of some drugs (antioxidants--asiaticoside, vitamin E and ascorbic acid; calcium D-pantothenate, exogenous fibronectin; antileprosy drugs--oil of hydnocarpus; alcoholic extract of yeast) accelerate wound healing. Thymic peptide thymosin beta 4 (T beta 4R) topically applicated, increases collagen deposition and angiogenesis and stimulates <em>keratinocyte</em> migration. Thymosin alpha 1 (T alpha 1R), peptide isolated from the thymus, is a potent chemoattractant which accelerates angiogenesis and wound healing. On the contrary, steroid drugs, hemorrhage and denervation of wounds have negative effect on the healing process.

Tumor necrosis <em>factor</em> (TNF)-like weak inducer of apoptosis (TWEAK) binds to its sole receptor fibroblast <em>growth</em> <em>factor</em>-inducible 14 (Fn14), participating in various inflammatory responses. Recently, TWEAK/Fn14 activation was found prominent in the lesions of cutaneous lupus erythematosus (CLE). This study was designed to further reveal the potential role of this pathway in Ro5<em>2</em>-mediated photosensitization. TWEAK, Fn14, and Ro5<em>2</em> were determined in the skin lesions of patients with CLE. Murine <em>keratinocytes</em> received ultraviolet B (UVB) irradiation or plus TWEAK stimulation and underwent detection for Ro5<em>2</em> and proinflammatory cytokines. The chemotaxis of J774.<em>2</em> macrophages was evaluated on TWEAK stimulation of cocultured <em>keratinocytes</em>. We found that TWEAK, Fn14, and downstream cytokines were highly expressed in CLE lesions that overexpressed Ro5<em>2</em>. Moreover, TWEAK enhanced the UVB-induced Ro5<em>2</em> upregulation in murine <em>keratinocytes</em>. Meanwhile, TWEAK stimulation of <em>keratinocytes</em> favored the migration of macrophages through promoting the production of chemokine C-C motif ligands 17 and <em>2</em><em>2</em>. Furthermore, Fn14 siRNA transfection or nuclear <em>factor</em>-kappa B (NF-κB) inhibitor abrogated the TWEAK enhancement of Ro5<em>2</em> expression in <em>keratinocytes</em>. Similarly, TNF receptor associated <em>factor</em> <em>2</em> (TRAF<em>2</em>) siRNA reduced the protein level of Ro5<em>2</em> in these cells upon TWEAK stimulation. Interestingly, UVB irradiation increased the expression of TNF receptor type 1 (TNFR1) but not affecting TNFR<em>2</em> expression in <em>keratinocytes</em>. In conclusion, the TWEAK/Fn14 signaling participates in Ro5<em>2</em>-mediated photosensitization and involves the activation of NF-κB pathway as well as the function of the TRAF<em>2</em>/TNFR partners.

Psoriasis, a chronic autoimmune-related skin disease, involves both immune and non-immune cells like T cells and <em>keratinocytes</em>. This study investigates the regulatory role of T cells-<em>keratinocyte</em> interactions during psoriasis on immune <em>factors</em> production. Cytokines and chemokines were evaluated by multiplex and ELISA assays in an in vitro model of co-culture of <em>keratinocytes</em> with T lymphocytes. <em>Keratinocytes</em> were from psoriatic skin lesions or healthy skin. T lymphocytes were from healthy volunteers. Psoriatic <em>keratinocytes</em> (PKs) alone generated concentrations of tumor necrosis <em>factor</em> (TNF)-α, interleukin (IL)-6, granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), IL-1β, IL-8, monocyte chemotactic protein (MCP)-1, interferon-γ-induced protein 10 kDa (IP-10) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) higher than those produced by healthy <em>keratinocytes</em> (HKs). In contrast, IL-1α and IL-Ra production was reduced in PKs. Normal T cells, which had no effect on HKs, increased the production of TNF-α, IL-6, GM-CSF, IL-8, MCP-1 and IP-10 by PKs, but did not influence PK production of IL-1β, IL-1α, IL-Ra and VEGF. The most striking effects were obtained with PK- and IL-<em>2</em>-stimulated T lymphocytes: most of the above cytokines and chemokines were greatly upregulated, except IL-1β and VEGF that were decreased or unchanged, respectively. In addition, fractalkine was overproduced in this latter condition only. Our results indicate (1) a functional interaction between <em>keratinocytes</em> and T lymphocytes that requires a direct cellular contact, and (<em>2</em>) a reciprocal influence that depends on cytokine and chemokine types. In conclusion, lesional <em>keratinocytes</em> from psoriasis vulgaris alter functions of normal T lymphocytes that conversely modulate these <em>keratinocytes</em>.

Transforming <em>growth</em> <em>factor</em> alpha (TGF(alpha)) stimulates type II alveolar epithelial cell proliferation and also is associated with fibrosis. We studied the changes in bronchoalveolar lavage (BAL) TGF(alpha) protein in a neonatal rabbit hyperoxia-fibrosis model (100% O(<em>2</em>) for 8 to 9 days, followed by 60% O(<em>2</em>) to 36 days of age). Hyperoxia increased TGF(alpha) protein and delayed the appearance of mature lower molecular weight (MW) TGF(alpha) isoforms at postnatal days 6 and 8 during the acute injury period. At 3 and 5 weeks, after chronic hyperoxia exposure, there was an increase in lower MW TGF(alpha) peptides during the fibrotic period. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is also a type II cell mitogen. In vitro studies of <em>keratinocytes</em> suggest that KGF-induced proliferation is mediated through TGF(alpha). Intratracheal KGF instillation into adult wild-type and TGF(alpha)-null mice demonstrated that the KGF induced equivalent robust levels of proliferation in both TGF(alpha) deficient and wild-genotype mice. In conclusion, there are both quantitative and qualitative changes in TGF(alpha) protein in a hyperoxia-induced fibrosis neonatal rabbit model during periods of type II cell proliferation and fibrosis.

Staurosporine is a potent but nonselective inhibitor of protein kinase C (PKC) and blocks responses to 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA) in several cell types in vitro. In cultured primary mouse <em>keratinocytes</em>, however, staurosporine fails to inhibit TPA-mediated <em>keratinocyte</em> maturation and itself elicits responses that are similar to TPA (T. Sako et al., Cancer Res., 48: 4646-4650, 1988). After exposure to 10 nM staurosporine for <em>2</em>4 h, essentially all <em>keratinocytes</em> undergo morphological differentiation, whereas 160 nM TPA induces this response in about 50% of epidermal cells. These concentrations of staurosporine and TPA cause a 4-5-fold induction of epidermal transglutaminase activity and cornified envelopes, both markers of the terminal stage of <em>keratinocyte</em> differentiation. Staurosporine, but not TPA, also induces morphological and biochemical maturation in <em>2</em> neoplastic mouse <em>keratinocyte</em> cell lines, 308 and SP-1. The ability of staurosporine to elicit the same responses as TPA suggested that it may be functioning paradoxically as a PKC agonist in intact <em>keratinocytes</em>. In support of this hypothesis, staurosporine induces ornithine decarboxylase activity, inhibits 1<em>2</em>5I-labeled epidermal <em>growth</em> <em>factor</em> binding, and induces expression of c-fos mRNA. Down-regulation of PKC by pretreatment of primary <em>keratinocytes</em> with 60 nM bryostatin partially blocks staurosporine-mediated induction of cornified envelopes and inhibition of 1<em>2</em>5I-labeled epidermal <em>growth</em> <em>factor</em> binding, implicating PKC in these responses. The ability of staurosporine to mimic and/or enhance certain responses to TPA suggests that this agent is acting as a functional PKC agonist in cultured <em>keratinocytes</em>.

In skin, the distribution of integrins is compartmentalized. Whereas the alpha 6 beta 4 integrin complex is polarized to the basal portion of proliferating cells in the basal layer juxtaposed to the basement membrane, alpha 3 beta 1 integrin receptors are localized on the cell surface surrounding basal and suprabasal cells, suggesting beta 1 integrins mediate both cell-matrix and cell-cell interactions. As initiation of maturation in skin is associated with the detachment of cells from the basement membrane, the early loss of alpha 6 beta 4, but not alpha 3 beta 1, integrin expression could be a determining <em>factor</em> in the transition from the proliferating to a differentiating <em>keratinocyte</em>. We have studied the regulation of adhesion potential and integrin expression during differentiation of mouse basal <em>keratinocytes</em> culture in 0.05 mM Ca<em>2</em>+ medium and induced to differentiate in 0.1<em>2</em> mM Ca<em>2</em>+ medium. Within 1<em>2</em>-<em>2</em>4 h after elevation of Ca<em>2</em>+, a selective loss of the alpha 6 beta 4 integrin complex is associated with the induction of the spinous cell marker keratin 1. This early differentiation phenotype coincides with loss of cell attachment mediated by alpha 6 beta 4 to laminins 1 and 5 but not a fibronectin or collagen IV. Selective loss of attachment to laminin is also detected in spinous cells isolated from newborn epidermis in vivo. The loss of alpha 6 and beta 4 protein expression is a consequence of transcriptional and posttranscriptional events, including reduction in mRNA transcripts, reduced synthesis of the alpha 6 protein, and enhanced processing of the alpha 6 and beta 4 chains as determined by Western blots and pulse-chase experiments in metabolically labeled <em>keratinocytes</em>. Selective processing of the beta 4 intracellular domain is detected before loss of beta 4 from the cell surface in basal <em>keratinocytes</em>, and this process is accelerated during differentiation. Whereas early <em>keratinocyte</em> maturation is linked to the selective loss of the alpha 6 beta 4 complex, loss of both beta 1 and beta 4 integrin mRNA and protein occurs as cells proceed to later stages in the differentiation program as induced by 0.5 mM Ca<em>2</em>+ or suspension culture. These conditions are characterized by accelerated expression of transglutaminase; reduced keratin 1 protein; loss of adhesion to fibronectin, laminin 1, laminin 5, and collagen IV; and rapid cell death. Contributing to the down-regulation of beta 1 integrins during terminal differentiation is a selective sensitivity of alpha 3 beta 1 but not alpha 6 beta 4 to down-regulation by transforming <em>growth</em> <em>factors</em> beta 1 and beta <em>2</em>, <em>factors</em> that are also expressed differentially in normal skin. This study indicates that down-regulation of the alpha 6 beta 4 but not beta 1 integrins occurs during the initial steps of <em>keratinocyte</em> differentiation and is associated with detachment from the laminin matrix. Such changes could contribute an important signal to initiate the process of terminal <em>keratinocyte</em> differentiation.

AP-<em>2</em>alpha and c-MYC are important transcription <em>factors</em> involved in multiple cellular processes. They each display the paradoxical capacities to stimulate both cell proliferation and apoptosis under different conditions. In the present study we found that over expression of c-MYC was associated with accumulation of reactive oxygen species (ROS) and apoptosis in human <em>keratinocytes</em>, both of which were significantly inhibited by co-expression of AP-<em>2</em>. The effects of AP-<em>2</em> on c-MYC were active at several levels. First, AP-<em>2</em> and c-MYC were confirmed to interact at the protein level as previously described. In addition, forced expression of AP-<em>2</em> significantly decreased steady state levels of c-MYC mRNA and protein. These findings suggested that AP-<em>2</em> may have a direct effect on the c-myc gene. Chromatin immunoprecipitation assays demonstrated that AP-<em>2</em> proteins bound to a cluster of AP-<em>2</em> binding sites located within a <em>2</em> kb upstream regulatory region of c-myc These results suggest that the negative regulation of AP-<em>2</em> on c-MYC activity was achieved through binding of AP-<em>2</em> protein to the c-myc gene. The effects of AP-<em>2</em> on c-MYC induced ROS accumulation and apoptosis in epidermal <em>keratinocytes</em> are likely to play an important role in cell <em>growth</em>, differentiation and carcinogenesis of the skin.

Fibrosis is associated with a variety of skin diseases and causes severe aesthetic and functional impairments. Functional studies in rodents, together with clinical observations, strongly suggest a crucial role of chronic injury and inflammation in the pathogenesis of fibrotic diseases. The phenotype of mice lacking fibroblast <em>growth</em> <em>factor</em> (FGF) receptors 1 and <em>2</em> in <em>keratinocytes</em> supports this concept. In these mice, a defect in <em>keratinocytes</em> alone initiated an inflammatory response, which in turn caused <em>keratinocyte</em> hyperproliferation and dermal fibrosis. As the mechanism underlying this phenotype, we identified a loss of FGF-induced expression of claudins and occludin, which caused abnormalities in tight junctions with concomitant deficits in epidermal barrier function. This resulted in severe transepidermal water loss and skin dryness. In turn, activation of <em>keratinocytes</em> and epidermal γδ T cells occurred, which produced IL-1 family member 8 and S100A8 and S100A9. These cytokines attracted immune cells and activated fibroblasts, resulting in a double paracrine loop through production of <em>keratinocyte</em> mitogens by dermal cells. In addition, a profibrotic response was induced in fibroblasts. Our results highlight the importance of an intact epidermal barrier for the prevention of inflammation and fibrosis and the role of chronic inflammation in the pathogenesis of fibrotic diseases.

The binding of 1<em>2</em>5I-labelled transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) to human alpha <em>2</em>-macroglobulin (alpha <em>2</em>M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha <em>2</em>M-methylamine and minimally, if at all, to native alpha <em>2</em>M. Preparations of alpha <em>2</em>M-proteinase complex were generated by incubating a standard concentration of alpha <em>2</em>M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-<em>2</em>.0 microM). The 1<em>2</em>5I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha <em>2</em>M, or r value, used to form the alpha <em>2</em>M-proteinase complex. With all three proteinases, r values of <em>2</em> or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha <em>2</em>M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse <em>keratinocytes</em> were consistent with the 1<em>2</em>5I-TGF-beta-binding experiments. alpha <em>2</em>M-trypsin and alpha <em>2</em>M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 1<em>2</em>5I-TGF-beta 1 binding to alpha <em>2</em>M-methylamine was at least 80% non-covalent. Reaction of alpha <em>2</em>M-methylamine with iodoacetamide or 5,5'-dithiobis-(<em>2</em>-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 1<em>2</em>5I-TGF-beta 1 to alpha <em>2</em>M-methylamine. Cleavage of the 'bait regions' in alpha <em>2</em>M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha <em>2</em>M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha <em>2</em>M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.

OBJECTIVE

Hydrogen sulfide ( H₂S ) is a volatile sulfur compound responsible for physiological halitosis. H₂S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H₂S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H₂S.

METHODS

OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H₂S for 24 and 48 h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR.

RESULTS

The levels of apoptosis were significantly increased following incubation in the presence of H₂S, especially after 48 h (36.95 ± 1.91% vs. 4.77 ± 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 ± 9.84 units/mL vs. 1.5 ± 0 units/mL and 32.22 ± 10.23 units/mL vs. 0.15 ± 0 units/mL, respectively, at 48 h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased.

CONCLUSIONS

Following incubation with H2 S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.

EKB-569 is an irreversible inhibitor of epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) tyrosine kinase. It inhibits EGF-induced phosphorylation of EGF-R and the <em>growth</em> of tumors that overexpress EGF-R in animal models. In clinical trials, EKB-569 and all other EGF-R inhibitors cause skin rashes. To understand the latter phenomenon, the effect of EKB-569 on EGF-R as well as downstream signaling to phosphoinositide 3-kinase-protein kinase B (AKT), extracellular signal-regulated kinase 1 and <em>2</em> (ERK1/<em>2</em>), or signal transducer and activator of transcription 3 (STAT3) pathways were compared in tumor cell lines and normal human <em>keratinocytes</em> (NHEK) grown in tissue culture. Tumor cell lines that have high (A431 epidermoid and MDA-468 breast carcinomas) and low (MCF-7 breast carcinoma) expression of EGF-R were used. NHEK cells express at least 15-fold less EGF-R than A431 cells. EKB-569 was a potent inhibitor of proliferation in NHEK, A431, and MDA-468 cells (IC(50) = 61, 1<em>2</em>5, and <em>2</em>60 nM, respectively) but not MCF-7 cells (IC(50) = 3600 nM). EKB-569 was also a potent inhibitor of EGF-induced phosphorylated EGF-R (pEGF-R) in A431 and NHEK cells (IC(50) = <em>2</em>0-80 nM). The reduction in pEGF-R paralleled inhibition of phosphotyrosine-705 STAT3, while the inhibition of phosphorylated AKT and phosphorylated ERK1/<em>2</em> occurred at higher concentrations of EKB-569 (75-500 nM) in both A431 and NHEK cells. The effects were specific because EKB-569 did not inhibit the nuclear <em>factor</em>-kappaB pathway. It is proposed that skin toxicity associated with EKB-569 is due to inhibition of EGF-R signaling. Downstream signal transduction markers, particularly the activation status of STAT3, may be useful surrogate markers to guide clinical development of EGF-R inhibitors.

BACKGROUND

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(<em>2</em>+) signaling, <em>growth</em>, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many <em>factors</em> have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK.

OBJECTIVE

Here we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca(<em>2</em>+) signaling.

METHODS

Fibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca(<em>2</em>+) signaling and fibroblast proliferation. Changes in intracellular Ca(<em>2</em>+) were detected by fluorescence with fura-<em>2</em>/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old).

RESULTS

K6PC-5 promoted fibroblast proliferation and procollagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca(<em>2</em>+) concentration ([Ca(<em>2</em>+)](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca(<em>2</em>+)](i), an effect independent of the classical PLC/IP(3)-mediated pathway. The K6PC-5-induced [Ca(<em>2</em>+)](i) oscillations were dependent on thapsigargin-sensitive Ca(<em>2</em>+) stores and Ca(<em>2</em>+) entry. Topical application of K6PC-5 for <em>2</em> weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function.

CONCLUSIONS

These results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging.

High altitude pulmonary oedema (HAPE) severely affects non-acclimatized individuals and is characterized by alveolar flooding with protein-rich oedema as a consequence of blood-gas barrier disruption. Limited choice for prophylactic treatment warrants effective therapy against HAPE. <em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>) has shown efficiency in preventing alveolar epithelial cell DNA damages in vitro. In the current study, the effects of KGF-<em>2</em> intratracheal instillation on mortality, lung liquid balance and lung histology were evaluated in our previously developed rat model of HAPE. We found that pre-treatment with KGF-<em>2</em> (5 mg/kg) significantly decreased mortality, improved oxygenation and reduced lung wet-to-dry weight ratio by preventing alveolar-capillary barrier disruption demonstrated by histological examination and increasing alveolar fluid clearance up to 150%. In addition, KGF-<em>2</em> significantly inhibited decrease of transendothelial permeability after exposure to hypoxia, accompanied by a 10-fold increase of Akt activity and inhibited apoptosis in human pulmonary microvascular endothelial cells, demonstrating attenuated endothelial apoptosis might contribute to reduction of endothelial permeability. These results showed the efficacy of KGF-<em>2</em> on inhibition of endothelial cell apoptosis, preservation of alveolar-capillary barrier integrity and promotion of pulmonary oedema absorption in HAPE. Thus, KGF-<em>2</em> may represent a potential drug candidate for the prevention of HAPE.

UV light is a potent stimulus for <em>keratinocytes</em> to release several cytokines. Recently, UV light was shown to inhibit <em>keratinocyte</em> release of IL-7, a <em>growth</em> <em>factor</em> for dendritic epidermal T cells. Since to date IL-7 is the only <em>keratinocyte</em>-derived cytokine down-regulated by UV light, we addressed the molecular mechanisms involved. IFN-gamma treatment of the murine <em>keratinocyte</em> cell line Pam <em>2</em>1<em>2</em> resulted in an up-regulation of IL-7 mRNA, while IL-7 transcripts were suppressed in cells exposed to UV before IFN-gamma. Because IFN-gamma induces IL-7 via activation of an IFN-stimulated response element (ISRE) located in the 5' upstream region of the IL-7 gene, bandshift assays were performed using the ISRE sequence from the IL-7 gene. Nuclear extracts from untreated cells revealed two bands, a slower migrating band identified by supershift analysis as IFN regulatory <em>factor</em>-<em>2</em> (IRF-<em>2</em>), a transcriptional repressor, and a more rapidly migrating band identified as IRF-1, a transcriptional activator. IFN-gamma significantly induced IRF-1 binding, whereas UV treatment plus IFN-gamma decreased IRF-1 binding, suggesting that UV light suppresses IFN-gamma-induced expression of IL-7 by interfering with IRF-1. Chloramphenicol transferase assay confirmed functional relevance, showing that the minimal promoter sequence for the ISRE explicitly responded to IFN-gamma, which was suppressed by UV irradiation. Northern blot analysis using an IRF-1 cDNA probe revealed that UV light reduced IFN-gamma-induced IRF-1 mRNA. This study demonstrates that UV light can inhibit cytokine activities by interference with transcriptional activators. This newly described ability of UV light may contribute to its immunosuppressive properties.

Members of the fibroblast <em>growth</em> <em>factor</em> (FGF) family are important <em>growth</em>-regulatory elements. Of the FGFs, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) appears to have unique properties that implicate it as a paracrine <em>factor</em> in the prostate. Two KGF transcripts (approximately to <em>2</em>.4 and 5.0 kb) encode a protein of approximately <em>2</em><em>2</em> kDa. In contrast to several other members of the FGF family, KGF has a signal peptide and is actively secreted. Cellular response to KGF is mediated by a specific receptor that is transcribed from an alternately spliced variant of the FGF type <em>2</em> receptor (FGFR-<em>2</em>). KGF transcripts have been detected in prostatic tissues and in stromal cells cultured from rat and human prostates as well as in a variety of stromal cells derived from other organs. Prostatic epithelial cells and numerous other types of epithelial cells are targets of KGF's mitogenic activity. Several <em>factors</em> involved in wound healing regulate the expression of KGF, but androgen regulation of KGF is of greatest relevance to the role of KGF in the prostate. Current efforts to localize and manipulate KGF activity in vivo should reveal the significance of KGF expression and function in the prostate and in other organs.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and its receptor are involved in various types of epithelial repair processes. To gain insight into the molecular mechanisms of KGF action in the healing skin wound, we searched for genes which are regulated by this <em>factor</em> in cultured <em>keratinocytes</em>. Using the PCR-select technology we constructed a subtractive cDNA library. One of the KGF-regulated genes that we identified was shown to encode caveolin-1, a major component of caveolar membranes. Caveolin-1 is involved in a wide variety of cellular processes, particularly in the regulation of various signal transduction pathways. Caveolin-1 mRNA levels increased in cultured <em>keratinocytes</em> after KGF treatment. By in situ hybridization and immunohistochemistry we found a strong expression of caveolin-1 in the KGF-responsive basal <em>keratinocytes</em> of the epidermis and the hyperproliferative epithelium of the wound as well as in endothelial cells and in other cells of the granulation tissue. In 13-day wounds expression of caveolin-1 mRNA was restricted to the regenerated dermis. In addition to caveolin-1, the mRNA expression of caveolin-<em>2</em>, a second member of the caveolin family, was also induced in <em>keratinocytes</em> after stimulation with KGF but also with other <em>growth</em> <em>factors</em> and cytokines. In contrast to caveolin-1, caveolin-<em>2</em> protein was expressed in all layers of the normal epidermis and in the suprabasal layers of the hyperproliferative wound epithelium. These results demonstrate a differential expression of caveolin-1 and -<em>2</em> in proliferating versus differentiating <em>keratinocytes</em>.