OBJECTIVE

Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway is constitutively activated in melanoma. AZD6<em>2</em>44 blocks MEK1/<em>2</em>, inhibiting ERK phosphorylation. We focus on associated cutaneous toxicity and we attempt to understand the underlying pathophysiology and design treatment strategies.

METHODS

Dermatologic conditions of <em>2</em><em>2</em> patients with unresectable melanoma stage III/IV in a phase II trial were evaluated. Thirteen patients received AZD6<em>2</em>44 initially, and nine patients were treated with AZD6<em>2</em>44 following tumor progression with temozolomide. Biopsies were compared with matched controls in normal skin. Immunohistochemistry was performed. Half-side treatment of acute skin toxicity compared therapeutic options.

RESULTS

Nineteen of <em>2</em><em>2</em> (86%) AZD6<em>2</em>44-treated patients presented with cutaneous eruptions. Seventeen patients (77%) developed acute papulopustular rash. Chronic skin changes included xerosis, paronychia, and fissured fingertips, resembling cutaneous toxicity of epidermal growth factor receptor inhibition. In addition, we observed reduced pigmentation of hair and skin. Histology of acute skin lesions revealed a significant increase of apoptotic keratinocytes (P = 0.0008), focal neutrophilic infiltrates, destruction of the adnexal structures by neutrophils, and reduced cytokeratins. A significant proliferation shift from basal to suprabasal keratinocytes was shown in acute and chronic lesions. The number and viability of melanocytes was not affected. Corticosteroids plus antibacterial topical therapy ameliorate acute skin toxicity.

CONCLUSIONS

AZD6<em>2</em>44-associated skin reactions partly overlap with those observed upon epidermal growth factor receptor inhibition. Additionally, pigmentation of skin and hair is affected. The interruption of the MEK signaling pathway results in an acute keratinocyte stress response with disturbed epidermal homeostasis, inflammation, and tissue damage. Chronic adaptation controls inflammatory tissue damage but leads to cutaneous malfunctions that explain chronic skin toxicity.

Psoriasis is characterized by alterations in both the epidermis and dermis of the skin. Epidermal <em>keratinocytes</em> display marked proliferative activation and differentiate along an "alternate" or "regenerative" pathway, while the dermis becomes infiltrated with leukocytes, particularly interleukin <em>2</em> (IL-<em>2</em>) receptor-bearing "activated" T cells. Psoralens, administered by the oral route, have therapeutic effects in psoriasis when photochemically activated by ultraviolet A light (PUVA therapy). Recently psoralen bath therapy has been introduced to more effectively deliver this agent to the diseased skin. We have correlated the efficacy of PUVA bath therapy with its effects on specific molecular and cellular parameters of disease, in 10 consecutive patients with recalcitrant psoriasis. Rapid clearing of lesions occurred in 8 out of 10 patients. Biopsies were taken from lesional and nonlesional skin before and after a single round of therapy, and observation was continued in our Clinical Research Center at The Rockefeller University. Enumeration of cycling <em>keratinocytes</em> with the Ki-67 monoclonal antibody showed that PUVA reduced cell proliferation by 73%. The pathological increase in insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) receptors was reversed, whereas epidermal <em>growth</em> <em>factor</em> (EGF) receptors, which are also increased in psoriasis, remained unchanged. <em>Keratinocyte</em> proteins that are expressed in abnormal sites of the epidermis during psoriasis, i.e., keratin 16, filaggrin, and involucrin, were, after PUVA treatment, localized to their normal sites. Epidermal and dermal T-lymphocytes (CD3+), as well as CD4+, CD8+, and IL-<em>2</em> receptor+ subsets, were strongly suppressed by PUVA, with virtual elimination of IL-<em>2</em> receptor+ T cells in some patients. Consistent with diminished lymphocyte activation, HLA-DR expression by epidermal <em>keratinocytes</em> was markedly reduced in treated skin. In comparison to cyclosporine treatment of psoriasis, PUVA therapy leads to more complete reversal of pathological epidermal and lymphocytic activation, changes which we propose to be the cellular basis for a more sustained remission of disease after PUVA treatment.

In order to validate a model for predictive screening of dermatological drugs, we used a customized cDNA macro-array system containing 475 skin-related genes to analyze the gene expression patterns in human <em>keratinocytes</em> from different origins: (1) normal human epidermal <em>keratinocyte</em> mono-layer cultures, (<em>2</em>) the commercially available SkinEthic reconstituted human epidermis model, and (3) biopsies of normal human epidermis. Few markers of those that were detected significantly in <em>keratinocyte</em> mono-layers or in reconstituted epidermis were undetected or detected at very low level in the normal epidermis biopsies. A comparative expression of more than 100 markers could be evidenced in both normal epidermis and reconstituted epidermis samples; however, only 90% of these were detected in <em>keratinocyte</em> mono-layers: expression of several terminal differentiation markers, such as filaggrin, loricrin, and corneodesmosin were strongly detected in normal epidermis and reconstituted epidermis, but were not significantly expressed in <em>keratinocyte</em> mono-layers. Under the experimental conditions described herein, the reconstituted human epidermis model was found to significantly reproduce the gene expression profile of normal human epidermis. Using the same methodology, we then investigated the effects of all-trans retinoic acid, 9-cis retinoic acid, all-trans retinol and a commercialized tretinoin-containing cream (Retacnyl) on the gene expression profiles of reconstituted human epidermis. According to the nature and the length of the treatments, more than 40 genes were found significantly modified. Among the genes whose expression was decreased, we found cytokeratins 1, 10, <em>2</em>E, and 6B, several cornified envelope precursors, integrins alpha 3, alpha 6, beta 1, beta 4, some components of desmosomes, of hemi-desmosomes and of the epidermal basement membrane. Transcriptional upregulation was observed for keratins 18 and 19, autocrine and paracrine <em>growth</em> <em>factors</em> such as HB-EGF, IGF 1, PDGF-A, calgranulins A and B, interleukin-1 alpha and the other IL-1-related markers, type II IL-1 receptor and type I IL-1-receptor antagonist. Our results confirm most of the known effects of retinoids on human epidermis, but also give new insights into their complex pharmacological activity on skin. The reconstituted human epidermis used proves to be a highly predictive model for efficacy evaluation of skin-targeted compounds, such as retinoids.

BACKGROUND

Hypoxia-inducible <em>factor</em> (HIF)-1α, part of the heterodimeric transcription <em>factor</em> that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic <em>growth</em> <em>factors</em>, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-<em>2</em> (PHD-<em>2</em>), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-<em>2</em> in the epidermis and dermis have not been clearly defined in wound healing.

METHODS

Epidermal and dermal specific PHD-<em>2</em> knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-<em>2</em> mice with heterozygous K14-Cre mice and heterozygous Col1A<em>2</em>-Cre-ER mice to get homozygous floxed PHD-<em>2</em>/heterozygous K14-Cre and homozygous floxed PHD-<em>2</em>/heterozygous floxed Col1A<em>2</em>-Cre-ER mice, respectively. Ten to twelve-week-old PHD-<em>2</em> KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.

RESULTS

qRT-PCR demonstrated a significant decrease of PHD-<em>2</em> in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-<em>2</em> KO mice relative to control mice (*p<0.05). PHD-<em>2</em> KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-<em>2</em> tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).

CONCLUSIONS

PHD-<em>2</em> plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-<em>2</em> KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic <em>factors</em> such as VEGF.

1alpha,<em>2</em>5-dihydroxyvitamin D(3) [1,<em>2</em>5(OH)(<em>2</em>)D(3)] regulates mineral homeostasis and exhibits potent anti-proliferative, prodifferentiative, and immunomodulatory activities. It mediates these effects by binding to the vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. As a result of <em>keratinocyte</em> differentiation and anti-proliferation activities, 1,<em>2</em>5(OH)(<em>2</em>)D(3) and its synthetic analogs are therapeutically effective in psoriasis and show promise for the treatment of actinic keratosis and squamous cell carcinoma. To elucidate the VDR signaling pathway in <em>keratinocytes</em>, we examined the gene expression profile with 1,<em>2</em>5(OH)(<em>2</em>)D(3) treatment using oligonucleotide microarrays. Out of the 1<em>2</em>,600 genes investigated, 8<em>2</em> were upregulated and 16 were downregulated and many of these were involved in differentiation, proliferation, and immune response. We have identified three vitamin D-responsive chromosomal loci (1p36, 19q13, and 6p<em>2</em>5) and show the induction of various class II tumor suppressor/<em>growth</em>-regulatory genes in response to 1,<em>2</em>5(OH)(<em>2</em>)D(3). Finally, quantitative differences in gene expression revealed a vitamin D-regulated differentiation network and identified peptidylarginine deiminases, kallikreins, serine proteinase inhibitor family members, Kruppel-like <em>factor</em> 4, and c-fos as vitamin D-responsive genes, whose protein products may play an important role in epidermal differentiation in normal and diseased state.

The level of expression of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) mRNA has been measured in human breast cell lines, purified populations of epithelial cells, myoepithelial cells and fibroblasts from reduction mammoplasty tissue and a panel of 4<em>2</em> breast cancers and 30 non-malignant human breast tissues using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. We found similar levels of KGF mRNA in malignant and non-malignant breast tissues. The study of the amount of KGF mRNA in breast cell lines and purified populations of cells revealed that fibroblasts are the predominant source of KGF with malignant and non-malignant epithelial cells containing very low levels of KGF mRNA. We have examined the distribution of fibroblast <em>growth</em> <em>factor</em> receptor (FGFR)-<em>2</em>-IIIb, which is a high-affinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-<em>2</em>-IIIb present on breast cancer cell lines was sufficient for KGF stimulation of breast cancer cell proliferation. Other members of the fibroblast <em>growth</em> <em>factor</em> family have been either not expressed in the human breast (FGF3, FGF4) or have been found at much reduced levels in breast cancer (FGF1, FGF<em>2</em>) and this is the first member of the family to potentially influence the progression of breast cancer through stimulation of cell division.

Keloid is a skin fibrotic disease with the characteristics of recurrence and invasion, its pathogenesis still remains unrevealed. The epithelial-mesenchymal transition (EMT) is critical for wound healing, fibrosis, recurrence, and invasion of cancer. We sought to investigate the EMT in keloid and the mechanism through which the EMT regulates keloid formation. In keloid tissues, the expressions of EMT-associated markers and transforming <em>growth</em> <em>factor</em> (TGF)-β1/Smad3 signaling were examined by immunohistochemistry. In the keloid epidermis and dermal tissue, the expressions of genes related to the regulation of skin homeostasis, fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) and p63, were analyzed using quantitative real-time polymerase chain reaction. The results showed that accompanying the loss of the epithelial marker E-cadherin and the gain of the mesenchymal markers fibroblast-specific protein 1 (FSP1) and vimentin in epithelial cells from epidermis and skin appendages, and in endothelial cells from dermal microvessels, enhanced TGF-β1 expression and Smad3 phosphorylation were noted in keloid tissues. Moreover, alternative splicing of the FGFR<em>2</em> gene switched the predominantly expressed isoform from FGFR<em>2</em>-IIIb to -IIIc, concomitant with the decreased expression of ΔNp63 and TAp63, which changes might partially account for abnormal epidermis and appendages in keloids. In addition, we found that TGF-β1-induced hair follicle outer root sheath <em>keratinocytes</em> (ORSKs) and normal skin epithelial cells underwent EMT in vitro with ORSKs exhibiting more obvious EMT changes and more similar expression profiles for EMT-associated and skin homeostasis-related genes as in keloid tissues, suggesting that ORSKs might play crucial roles in the EMT in keloids. Our study provided insights into the molecular mechanisms mediating the EMT pathogenesis of keloids.

Psoriasis is a chronic disease characterized by abnormal epidermal proliferation, inflammation and angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is overexpressed in psoriatic epidermis and that the level of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. We hypothesized that CGRP might regulate the expression of VEGF by human <em>keratinocytes</em>. VEGF expression in the CGRP-treated human <em>keratinocytes</em> was investigated and the CGRP signaling pathways were examined with respect to VEGF expression. The mRNA and protein levels of VEGF by CGRP were increased in a concentration-dependent manner. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor PD98059. The CGRP-mediated VEGF induction was also effectively inhibited by a pretreatment with the CGRP receptor antagonist CGRP 8-37. In addition, CGRP treatment induced rapid phosphorylation of ERK1/<em>2</em>, PD98059 and CGRP 8-37 were able to inhibit CGRP-induced ERK1/<em>2</em> phosphorylation. These results suggest that CGRP regulates the expression of VEGF through the CGRP receptor and ERK1/<em>2</em> MAPK signaling pathway in human HaCaT <em>keratinocytes</em>.

The forkhead box O (FOXO) family has been extensively investigated in aging and metabolism, but its role in tissue-repair processes remains largely unknown. Herein, we clarify the molecular aspect of the FOXO family in skin wound healing. We demonstrated that Foxo1 and Foxo3a were both up-regulated during murine skin wound healing. Partial knockout of Foxo1 in Foxo1(+/-) mice throughout the body led to accelerated skin wound healing with enhanced <em>keratinocyte</em> migration, reduced granulation tissue formation, and decreased collagen density, accompanied by an attenuated inflammatory response, but we observed no wound phenotype in Foxo3a(-/-) mice. Fibroblast <em>growth</em> <em>factor</em> <em>2</em>, adiponectin, and notch1 genes were significantly increased at wound sites in Foxo1(+/-) mice, along with markedly altered extracellular signal-regulated kinase 1/<em>2</em> and AKT phosphorylation. Similarly, transient knockdown of Foxo1 at the wound site by local delivery of antisense oligodeoxynucleotides enhanced skin wound healing. The link between FOXO1 and scarring extends to patients, in particular keloid scars, where we see FOXO1 expression markedly increased in fibroblasts and inflammatory cells within the otherwise normal dermis. This occurs in the immediate vicinity of the keloid by comparison to the center of the mature keloid, indicating that FOXO1 is associated with the over<em>growth</em> of this fibrotic response into adjacent normal skin. Overall, our data indicate that molecular targeting of FOXO1 may improve the quality of healing and reduce pathological scarring.

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(<em>2</em>)O(<em>2</em>)) formation, we analysed H(<em>2</em>)O(<em>2</em>) release in human HaCaT <em>keratinocytes</em> in response to lysophosphatidic acid (LPA), a mitogen for <em>keratinocytes</em>. LPA rapidly increased H(<em>2</em>)O(<em>2</em>) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(<em>2</em>)O(<em>2</em>) release was independent of the tyrosine kinase activity of the epidermal <em>growth</em> <em>factor</em> (EGF) receptor. Calcium chelators, phospholipase A(<em>2</em>) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(<em>2</em>)O(<em>2</em>) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(<em>2</em>)O(<em>2</em>) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(<em>2</em>)O(<em>2</em>) release in HaCaT cells.

The fibroblast <em>growth</em> <em>factors</em> (FGFs) act through high affinity tyrosine kinase receptors and, in addition, interact with lower affinity receptors that represent cell- or matrix-associated heparan sulfate proteoglycans. These lower affinity receptors modulate the biological activities of FGFs, but the mechanism by which they exert these effects is rather controversial. We have previously shown (Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. <em>2</em>68, <em>2</em>984-<em>2</em>988) that heparin potentiates the mitogenic activity of acidic FGF (aFGF) but inhibits that of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) in cells that express the KGF receptor (KGFR). Both <em>growth</em> <em>factors</em> bind the KGFR with high affinity. To gain an insight into the mechanism by which heparin modulates the biological activity of aFGF and KGF, we studied the effect of heparin and cell-associated heparan sulfates on the binding of these two <em>growth</em> <em>factors</em> to the KGFR. To work in a well defined system, we expressed functional KGFR in L6E9 myoblasts that lack detectable high affinity binding sites for FGFs. Low concentrations of heparin inhibited the binding of KGF to the KGFR. By contrast, similar concentrations of heparin enhanced the binding of aFGF to this receptor. The effect of heparin was not unique to L6E9 cells expressing the KGFR; it was also observed in Balb/MK cells that naturally express KGFR. Treatment of cells with sodium chlorate, which blocks sulfation of proteoglycans, reduced the binding of aFGF to its low and high affinity binding sites by 95 and 80%, respectively. In contrast, the binding of KGF to its high affinity binding sites was enhanced about <em>2</em>-fold. Similar results were obtained after degradation of cell-associated heparan sulfates by heparinase and heparitinase. Heparin restored the high affinity binding of aFGF to chlorate-treated cells and completely abolished the high affinity binding of KGF. Binding competition experiments suggest that aFGF and KGF bind to the same population of cell-associated heparan sulfates. In addition, KGF is apparently interacting with an as yet unidentified type of low affinity binding site that is not affected by chlorate or heparan sulfate-degrading enzymes. An important property of the FGF high affinity receptors is their ability to bind more than one ligand with high affinity. Based on the differential effect of cell-associated heparan sulfates on the binding of KGF and aFGF to the KGFR, we propose a regulatory role for cell-associated heparan sulfates as coordinators of the interaction of aFGF and KGF with the KGFR.(ABSTRACT TRUNCATED AT 400 WORDS)

The neu (c-erbB-<em>2</em>) gene encodes a transmembrane protein with tyrosine kinase activity that appears to be a <em>growth</em> <em>factor</em> receptor. Antibody was generated by immunization of rabbits with a synthetic polypeptide that was based on an internal sequence at the carboxy terminus of the molecule. This antibody was used to survey the expression of neu in human skin by immunohistochemistry. Significant protein was found in the squamous cell layer of the surface epidermis, in squamous cell carcinomas, in the external root sheath of hair follicles, and in eccrine gland secretory cells; it was poorly expressed in the basal cell layer and in a basal cell carcinomas. Increased neu expression appears to be associated with the differentiation of <em>keratinocytes</em>.

Heparin-binding EGF-like <em>growth</em> <em>factor</em> has been identified in human burn-wound fluid and in the epithelial cells of excised human partial-thickness burns. In the present study, the effect of heparin-binding EGF-like <em>growth</em> <em>factor</em> on burn-wound healing was evaluated by incorporating purified, recombinant heparin-binding EGF-like <em>growth</em> <em>factor</em> into slow-release cholesterol-lecithin pellets that were applied topically to partial-thickness burns in mice. Both experimental (heparin-binding EGF-like <em>growth</em> <em>factor</em>-treated) and control (untreated) mice were sacrificed on days 3, 5, and 10 after burn. Total burn-wound area, histology, <em>keratinocyte</em> proliferation, and in situ hybridization analysis for transforming <em>growth</em> <em>factor</em>-alpha were determined for each wound. The mean wound area of the experimental group on day 5 after burn was 1.07 cm<em>2</em>, compared with <em>2</em>.<em>2</em>0 cm<em>2</em> for controls (p=0.04). Cellular proliferation (as measured by immunohistochemical detection of 5-Bromo-<em>2</em>-deoxyuridine) on day 5 after burn in marginal <em>keratinocytes</em> and follicular epithelial cells was greater in the experimental group than in the control group. In situ hybridization showed up-regulation of transforming <em>growth</em> <em>factor</em>-alpha mRNA levels in experimental animals by day 5 after burn. Topical application of heparin-binding EGF-like <em>growth</em> <em>factor</em> significantly accelerates the reepithelialization of murine partial-thickness burns, increases <em>keratinocyte</em> proliferative activity, and enhances production of endogenous transforming <em>growth</em> <em>factor</em>-alpha mRNA.

Hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) <em>2</em>.<em>2</em>-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human <em>keratinocytes</em>, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p4<em>2</em>/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp <em>2</em> to dp <em>2</em>4 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.

We have previously developed an in vitro tumor progression model with mouse skin <em>keratinocytes</em> to study the molecular targets that mediate the tumor cell's progression from a benign to a malignant phenotype. The malignantly transformed cells were found to have elevated MAP kinase signaling and increases in AP-1, NFkappaB and cAMP response element (CRE) transcription <em>factors</em> activities compared to their benign counter-part. In this study, we showed that Rac1, a member of the Rho superfamily of small GTPases, functions as a key signaling molecule that mediates these malignant phenotypes. We used a doxycycline inducible system to express dominant negative Rac1 (N17 Rac1) in the squamous cell carcinomas producing 6M90 cell line. Conditional expression of the N17 Rac1 was able to decrease multiple markers of malignancy including: <em>growth</em> rate, colony formation, migration, invasion and most importantly, in vivo tumor <em>growth</em>. In addition, these phenotypic changes were accompanied by decreases in mitogenic signals, which include ERK1/<em>2</em>, JNK, and PI-3 kinase/Akt activation. Transactivation mediated by AP-1, NFkappaB, and CRE were also attenuated by expression of dominant negative Rac1. These observations led us to conclude that Rac1 signaling is required for the malignant phenotypes of the squamous cell carcinoma cells.

Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.5<em>2</em> pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of <em>2</em>.50-47.6<em>2</em> pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.<em>2</em> pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of <em>2</em>0.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(<em>2</em>). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of <em>2</em>.<em>2</em>-9.3 pg/mug) was similar to that of controls at baseline (<em>2</em>.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.

In this work, one of Zingiber officinale components, 10-shogaol, was tested with 1,1-diphenyl-<em>2</em>-picrylhydrazyl (DPPH) radical scavenging, metal chelating ability, and reducing power to show antioxidant activity. 10-Shogaol promoted human normal epidermal <em>keratinocytes</em> and dermal fibroblasts cell <em>growths</em>. 10-Shogaol enhanced <em>growth</em> <em>factor</em> production in transforming <em>growth</em> <em>factor</em>-β (TGF-β), platelet derived <em>growth</em> <em>factor</em>-αβ (PDGF-αβ) and vascular endothelial <em>growth</em> <em>factors</em> (VEGF) of both cells. In the in vitro wound healing assay for 1<em>2</em> or <em>2</em>4 h, with 10-shogaol, the fibroblasts and <em>keratinocytes</em> migrated more rapidly than the vehicle control group. Thus, this study substantiates the target compound, 10-shogaol, as an antioxidant for human skin cell <em>growth</em> and a migration enhancer with potential to be a novel wound repair agent.

Degradation of matrix proteins that constitute the dermal-epidermal junction and dermis by proteolytic enzymes is an essential step of melanoma invasion and metastasis, and this is primarily achieved by the matrix metalloproteinases. In this report, using zymography, we compared the basal secretion levels of active matrix metalloproteinase-<em>2</em> and matrix metalloproteinase-9 to levels in response to various extracellular matrix proteins, cytokines, and <em>growth</em> <em>factors</em> in normal human melanocyte cells and melanoma cell lines from different stages of neoplastic progression. Basal matrix metalloproteinase-9 activity was only detected in vertical <em>growth</em> phase and metastatic melanoma cell lines, suggesting that matrix metalloproteinase-9 is a candidate biomarker for identifying vertical <em>growth</em> phase and metastatic melanomas. Most melanoma cell lines and cultured normal melanocytes produced high levels of matrix metalloproteinase-<em>2</em>. In addition, both tumor necrosis <em>factor</em>-alpha and interleukin-1beta are strong inducers of active matrix metalloproteinase-9 in vertical <em>growth</em> phase melanoma cell lines, indicating a possible role of these cytokines in the switch from radial <em>growth</em> phase to vertical <em>growth</em> phase. We propose that these proinflammatory cytokines promote melanoma invasion in part through upregulating matrix metalloproteinase-9. Both these cytokines are released from <em>keratinocytes</em> in the epidermis by ultraviolet radiation. Thus, our study suggests that the microenvironment of melanoma cells is an important feature in melanoma progression, and ultraviolet-radiation-induced cytokines might promote the progression of melanoma through the release or activation of matrix metalloproteinases.

The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)-eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)-inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/<em>2</em> (ERK1/<em>2</em>) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the <em>growth</em> of premalignant and malignant <em>keratinocytes</em> more than the <em>growth</em> of normal counterparts by a combination of cell cycle arrest and apoptosis. The <em>growth</em> inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal <em>keratinocytes</em>, by both n-3 PUFAs was associated with epidermal <em>growth</em> <em>factor</em> receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/<em>2</em> and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/<em>2</em> phosphorylation, and the blocking antibody partially antagonized <em>growth</em> inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal <em>keratinocytes</em>. Our results show that, in part, EPA specifically inhibits SCC <em>growth</em> and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic <em>keratinocytes</em> to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/<em>2</em> phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo.

Epidermal cell proliferation is required for re-epithelialization during wound repair. Re-epithelialization of partial thickness excisional wounds in pigs is complete by 6 days after injury. The presence of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and heparin-binding molecules that are mitogenic for <em>keratinocytes</em> was examined in wound fluid obtained daily from these wounds. Two significant heparin-binding <em>growth</em> <em>factor</em> activities for Balb/MK <em>keratinocytes</em> were detected, a major one that was eluted from a heparin affinity column with 1.1 M NaCl and a minor one with 0.5 M NaCl. These activities appeared 1 day after injury, were maximal by <em>2</em>-3 days later, and disappeared by 6 days after injury. The molecule eluting with 1.1 M NaCl was heparin-binding EGF-like (HB-EGF). The levels of IGF-I in wound fluid were 45-90 ng/ml during the first 3 days following injury, decreased thereafter, and were not detectable 6 days after injury. IGF-I at 100 ng/ml, increased HB-EGF mitogenic activity for Balb/MK <em>keratinocytes</em> by 40-50 fold. We conclude that the synergism between IGF-I and HB-EGF and their relative concentration at the various days after injury may be important variables for regulating re-epithelialization during wound repair.

Cell culture conditions for the selective <em>growth</em> and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of <em>2</em>% fetal bovine serum, the human melanocyte cell culture environment contains the following <em>growth</em> <em>factor</em> supplements: epidermal <em>growth</em> <em>factor</em> (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve <em>growth</em> <em>factor</em> (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal <em>keratinocytes</em> and melanocytes for attachment to fibronectin, while the <em>growth</em> of the human melanocyte has been attributed to the mitogenic activity of the <em>growth</em> <em>factor</em>-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The <em>growth</em> <em>factor</em>-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte <em>growth</em> <em>factor</em> is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the <em>growth</em> of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.

BACKGROUND

Loss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie<em>2</em> system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie<em>2</em> system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie<em>2</em> receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation.

METHODS

Mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH<em>2</em>O ('low' tidal volume ∼7.5 ml/kg; LVT) or 18 cmH<em>2</em>O ('high' tidal volume ∼15 ml/kg; HVT). At initiation of HVT-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls.

RESULTS

HVT-ventilation influenced the Ang-Tie<em>2</em> system in lungs of healthy mice since Ang-1, Ang-<em>2</em> and Tie<em>2</em> mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie<em>2</em> signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of <em>keratinocyte</em>-derived chemokine (KC), macrophage inflammatory protein (MIP)-<em>2</em>, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HVT-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HVT-ventilated mice despite inhibition of inflammation, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and Ang-<em>2</em> expression.

CONCLUSIONS

Ang-1 treatment downregulates pulmonary inflammation, VEGF and Ang-<em>2</em> expression but does not protect against vascular leakage and impaired gas exchange induced by HVT-ventilation.

BACKGROUND

Astragaloside IV is the chief ingredient of Radix Astragali, which has been used in the Traditional Chinese Medicine as a major component of many polyherbal formulations for the repair and regeneration of injured organ and tissues. This study is to investigate the influence of astragaloside IV on both of the wound healing and scar formation.

METHODS

For the in vitro evaluation, the influence of the astragaloside IV in the wound scratch test of keratinocytes and the secretion of transforming growth factor-β1, a key factor contributing to scar formation were determined. With the rat skin excision model, the in vivo regulation of astragaloside IV on wound closure, angiogenesis and collagen disposition were also evaluated.

RESULTS

Astragaloside IV was shown to significantly promote the migration of keratinocytes in wound scratching assay. The superior effect of Astragaloside IV was observed at 100 μmol/L, in which the recover rates was increased with 2 and 3 folds after 48 h and 96 h respectively than that of blank control (P<0.01). Animal skin closure measurement showed that astragaloside IV could stimulate the wound healing, e.g. with 21% recover in contrast to the 8% of blank control at the 6th day. Biomechanic and Masson's trichrome stain analysis indicated that astragaloside IV may improve the strength of the repaired skin and promoted the angiogenesis and collagen synthesis. Meanwhile, the picrosirius-sirus red stain and Elisa test definitely showed the anti-scar effects of astragaloside IV by decreasing the levels of collagen I/III and TGF-β1 secretion by firbroblasts with a dose-dependent manner (25-100 μmol/L).

CONCLUSIONS

Astragaloside IV was shown a promising natural product with both healing and anti-scar effects for wound treatment. These results give the evidence for the application of astragaloside IV in the treatment of injury.

The <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF/FGF7), produced by stromal cells, is a key paracrine mediator of epithelial proliferation, differentiation and migration. Expression of the <em>growth</em> <em>factor</em> is increased in wound healing and in hyperproliferative epithelial diseases, as a consequence of the activation of dermal fibroblasts by the inflammatory microenvironment. The middle ear cholesteatoma, an aural epidermal pathology characterized by <em>keratinocyte</em> hyperproliferation and chronic inflammation, may represent a model condition to study the epithelial-mesenchymal interactions. To develop an in vitro model for this disease, we isolated and characterized human primary cultures of fibroblasts associated with the cholesteatoma lesion, analyzing their secretory behaviour and degree of differentiation or activation. Compared to the perilesional or control normal fibroblasts, all cultures derived from cholesteatoma tissues were less proliferating and more differentiated and their highly variable activated phenotype correlated with the secretion of KGF as well as of metalloproteases <em>2</em> and 9. Culture supernatants collected from the cholesteatoma-associated fibroblasts were able to increase the proliferation and differentiation of human <em>keratinocytes</em> assessed by the expression of Ki67 and keratin-1 markers. The single crucial contribution of the KGF released by fibroblasts on the <em>keratinocyte</em> biological response was shown by the specific, although partial, block induced by inhibiting the KGF receptor or by immunoneutralizing the <em>growth</em> <em>factor</em>. Altogether, these results suggest that the activation of the stromal fibroblasts present in the pathological tissue, and the consequent increased secretion of KGF, play a crucial role in the deregulation of the epidermal proliferation and differentiation.