Motivated by the need for bioactive materials that can accelerate dermal wound healing, this work describes the responses of <em>keratinocytes</em> to covalently immobilized epidermal <em>growth</em> <em>factor</em> (EGF) and how differences in the physical presentation of this <em>growth</em> <em>factor</em> affect cell function. Specifically, human <em>keratinocytes</em> were cultured with EGF delivered in soluble form, immobilized in a homogeneous pattern or immobilized in a gradient pattern, followed by analysis of cellular signaling, proliferation and migration. By changing the manner in which EGF was presented, <em>keratinocyte</em> behavior was dramatically altered. <em>Keratinocytes</em> responded to immobilized EGF patterns with high EGF receptor (EGFR) but low ERK1/<em>2</em> and Akt phosphorylation, accompanied by low proliferation, high migratory activity and coordinated cell alignment. In contrast, <em>keratinocytes</em> treated with soluble EGF experienced lower EGFR but higher ERK1/<em>2</em> and Akt phosphorylation and displayed a highly proliferative, rather than migratory, phenotype. <em>Keratinocytes</em> also responded to differences in immobilized EGF patterns, as migration was fastest upon immobilized gradients of EGF. A better understanding the interaction of cells with soluble vs. immobilized <em>growth</em> <em>factors</em> can help elucidate native healing events and achieve greater control over cell function, which may be useful in the development of wound repair treatments for many types of tissues.

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the <em>growth</em> <em>factors</em> such as epidermal <em>growth</em> <em>factor</em> and hepatocyte <em>growth</em> <em>factor</em> (HGF), but not <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., <em>2</em>005. Epidermal and hepatocyte <em>growth</em> <em>factors</em>, but not <em>keratinocyte</em> <em>growth</em> <em>factor</em>, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. <em>2</em>80, 7917--9<em>2</em>4]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(<em>2</em>+) for its activation, PKEvarepsilon is Ca(<em>2</em>+) independent. However, <em>growth</em> <em>factor</em>-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this <em>growth</em> <em>factor</em> was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.

The topical application of minoxidil may achieve millimolar concentrations in the skin. We investigated whether millimolar minoxidil could induce vascular endothelial <em>growth</em> <em>factor</em> (VEGF), a possible effector for minoxidil-mediated hair <em>growth</em>, and how it occurred at the molecular level. Cell-based experiments were performed to investigate a molecular mechanism underlying the millimolar minoxidil induction of VEGF. The inhibitory effect of minoxidil on hypoxia-inducible <em>factor</em> (HIF) prolyl hydroxylase-<em>2</em> (PHD-<em>2</em>) was tested by an in vitro von Hippel-Lindau protein (VHL) binding assay. To examine the angiogenic potential of millimolar minoxidil, a chorioallantoic membrane (CAM) assay was used. In human <em>keratinocytes</em> and dermal papilla cells, millimolar minoxidil increased the secretion of VEGF, which was not attenuated by a specific adenosine receptor antagonist that inhibits the micromolar minoxidil induction of VEGF. Millimolar minoxidil induced hypoxia-inducible <em>factor</em>-1α (HIF-1α), and the induction of VEGF was dependent on HIF-1. Moreover, minoxidil applied to the dorsal area of mice increased HIF-1α and VEGF in the skin. In an in vitro VHL binding assay, minoxidil directly inhibited PHD-<em>2</em>, thus preventing the hydroxylation of cellular HIF-1α and VHL-dependent proteasome degradation and resulting in the stabilization of HIF-1α protein. Minoxidil inhibition of PHD-<em>2</em> was reversed by ascorbate, a co<em>factor</em> of PHD-<em>2</em>, and the minoxidil induction of cellular HIF-1α was abrogated by the co<em>factor</em>. Millimolar minoxidil promoted angiogenesis in the CAM assay, an in vivo angiogenic test, and this was nullified by the specific inhibition of VEGF. Our data demonstrate that PHD may be the molecular target for millimolar minoxidil-mediated VEGF induction via HIF-1.

Matrix metalloproteinase-<em>2</em>8 (MMP-<em>2</em>8, epilysin) is highly expressed in the skin by <em>keratinocytes</em>, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-<em>2</em>8 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming <em>growth</em> <em>factor</em> beta. We recently reported the expression of MMP-<em>2</em>8 in both cartilage and synovium where expression is increased in patients with osteoarthritis. In human chondrosarcoma cells MMP-<em>2</em>8 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-<em>2</em>8 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-<em>2</em>8 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-<em>2</em>8 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-<em>2</em>8, but not catalytically inactive EA mutant increased the expression and activity of MMP-<em>2</em>, and all forms of MMP-<em>2</em>8 tested increased expression of MMP19 and TIMP3 mRNA. These data demonstrate that expression of MMP<em>2</em>8 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-<em>2</em>8 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous <em>keratinocytes</em>. Conventionally, the <em>keratinocytes</em> are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if <em>keratinocytes</em> could be expanded in culture without the need of a feeder layer and FCS. <em>Keratinocytes</em> were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. <em>Keratinocytes</em> in different passages (P<em>2</em>, P4, and P6) and freshly isolated cells were studied. <em>Keratinocytes</em> grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P<em>2</em>, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 <em>keratinocytes</em> grown on collagen type IV in P4 were needed to form 1 cm<em>2</em> epidermis, whereas 150,000 of freshly isolated <em>keratinocytes</em> were necessary. Using this culture technique sufficient numbers of <em>keratinocytes</em>, isolated from 1 cm<em>2</em> skin, were obtained to cover 400 cm<em>2</em> of wound surface in <em>2</em> weeks. The results show that <em>keratinocytes</em> can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Lysophosphatidic acid is a phospholipid <em>growth</em> <em>factor</em> and intercellular signaling molecule released by activated platelets and injured fibroblasts that affects <em>keratinocytes</em>, fibroblasts, neutrophils,and monocytes. We therefore hypothesized that lysophosphatidic acid could influence the inflammation and reepithelialization phases of wound healing. Lysophosphatidic acid (100 microM) was applied daily for 5 days to <em>2</em> mm-diameter excisional mouse ear skin wounds and re-epithelialization was measured. We also evaluated whether the bioactivity of lysophosphatidic acid could be increased by zinc (Zn<em>2</em>+, 1 mM). Inflammatory cells were counted in wound sections after 1, 3, or 5 days of healing. Reepithelialization was accelerated significantly by either lysophosphatidic acid or lysophosphatidic acid + Zn<em>2</em>+ (p < 0.01 and p < 0.0001, respectively). Both lysophosphatidic acid solutions significantly increased the amount of new epithelium in the wounds on days 1, <em>2</em>, and 3 (p < 0.05). Little wound area remained on day 4, and all wounds were fully reepithelialized by day 5. Lysophosphatidic acid did not affect the number of neutrophils or macrophages present in the wound area. Our findings show that lysophosphatidic acid increased the amount of reepithelialization in the early stages of cutaneous wound healing in excisional ear wounds, without affecting inflammatory function.

Expression of nNOS mRNA was found in normal human and mouse skin tissue. Upon wounding, we observed a rapid downregulation of nNOS mRNA and protein in wounds of mice; however, when repair continued, nNOS mRNA was strongly upregulated and nNOS protein expression peaked at late stages of healing. Immunohistochemistry revealed wound <em>keratinocytes</em> as the cellular source of nNOS. In line with the in vivo situation, we found a basal expression of nNOS in the human <em>keratinocyte</em> cell line HaCaT. A marked stimulation of nNOS expression in the cells was achieved with epidermal <em>growth</em> <em>factor</em> receptor (EGFR) ligands such as epidermal <em>growth</em> <em>factor</em> (EGF), heparin-binding EGF, transforming <em>growth</em> <em>factor</em>-alpha and two alternate splicing forms of the neuregulin gene. EGF-induced induction of nNOS was completely inhibited by the specific EGFR antagonist PD153035 and by the EGFR and Janus kinase <em>2</em>/3 inhibitor AG490. Activation of EGFR might contribute to the observed upregulation of nNOS also in skin repair, as we found a spatial and temporal correlation of phosphorylated EGFR (Y1173) with nNOS expression at the wound site. Thus, in addition to the inducible- and endothelial-type NOS isoforms, also nNOS expression is regulated in the process of cutaneous wound repair.

Human epidermis marks the interface between internal and external environments with the major task being to maintain body hydration. Alternating exposure of skin to a dry or humid environment is likely to cause changes in the epidermal water gradient resulting in osmotic alterations of epidermal <em>keratinocytes</em>. The present in vitro approach studied the effect of hypotonicity on cell-cell contact. It was demonstrated that hypotonic stress applied to human epithelial cells (HaCaT, A-431) induced upregulation of E-cadherin at both, the protein and mRNA level. 5'-deletional mutants of the E-cadherin promoter identified an element ranging from -53 to +31 that conveyed strong transactivation under hypotonic stress. In order to define relevant upstream regulators members of the MAP kinase family, the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and protein kinase B/Akt (PKB/Akt) were investigated. Hypotonic conditions led to a fast activation of ERK1/<em>2</em>, SAPK/JNK, p38, EGFR and PKB/Akt with distinct activation patterns. Experiments using specific inhibitors showed that p38 contributes to the E-cadherin transactivation under hypotonic conditions. Further upstream, adhesion was found to be a prerequisite for E-cadherin transactivation in this model. In summary, the present study provides evidence that E-cadherin is an osmo-sensitive gene that responds to hypotonic stress. The function of this regulation may be found in morphological changes induced by cell swelling. It is likely that induction of E-cadherin contributes to the stabilization between adjacent cells in order to withstand the physical forces induced by hypotonicity.

Incidence of melanoma continues to rise, and a better understanding of its genetics will be critical to improve diagnosis and develop new treatments. Here, we search for novel melanoma-specific genes that may serve as biomarkers and therapeutic targets by using an in vitro genetic screen. One identified cDNA encoded TROY, a member of the tumor necrosis <em>factor</em> receptor superfamily (TNFRSF). TROY is widely expressed during embryogenesis, but in adults expression is restricted to hair follicles and brain. However, TROY had never been associated with melanoma, and it was selected for further study. First we show that expression in melanoma is specific by semiquantitative RT-PCR analysis of a large panel of established tumor cell lines. Next, specificity of expression was evaluated by immunohistochemistry analysis of primary cell cultures and patient tissues. TROY is expressed in <em>2</em>/<em>2</em> primary melanoma cells and 45/45 melanoma tissue samples (p < 0.0001). With the exception of sebaceous glands, TROY is not expressed in normal skin biopsies (p < 0.0001) or primary skin cell cultures that contain <em>keratinocytes</em> and epidermal melanocytes, nor is it expressed in other skin tumor cells (p < 0.0001). Finally, we show that TROY regulates melanoma <em>growth</em>, because replication of melanoma cells with reduced TROY levels through treatment with short-interfering RNA was significantly decreased relative to control cells (p < 0.004). In summary, TROY is the first TNFRSF member that is a biomarker for melanoma. TROY also presents a potentially novel cell surface signaling target for inhibitors, cell and/or antibody-based immunotherapies.

OBJECTIVE

To investigate the effect of pretreatment with keratinocyte growth factor on acute permeability pulmonary edema.

METHODS

Prospective, randomized, controlled animal study.

METHODS

University research laboratory.

METHODS

Specific pathogen-free Sprague-Dawley rats.

METHODS

Acute permeability pulmonary edema was induced with an injection of alpha-naphthylthiourea, and lung leak was assessed in an isolated perfused lung model over 180 mins. Leak was confirmed with wet/dry lung weight ratios, and the alveolar fluid protein concentration was measured after bronchoalveolar lavage. The effect of pretreatment with keratinocyte growth factor (injected intratracheally 48 hrs before the experiment) on alpha-naphthylthiourea-induced pulmonary edema was assessed (keratinocyte growth factor/alpha-naphthylthiourea group). Control groups (Control and keratinocyte growth factor/Control) were also studied. Histopathology was performed for each of the four groups.

RESULTS

The alpha-naphthylthiourea produced an acute permeability pulmonary edema detected by lung leak over the 180-min ex vivo period of monitoring the isolated perfused lung (leak = 8+/-mL; wet/dry weight ratio 14.7+/-2; lavage protein 3.1+/-1 mg/mL). Pretreatment with keratinocyte growth factor significantly attenuated these parameters (leak = 2.3+/-0.4 mL; wet/dry weight ratio 7.1 +/- 0.5; lavage protein 0.28 +/-0.03 mg/mL), which were not significantly different from the control group and the keratinocyte growth factor/control group. Histopathology showed abundant type II pneumocyte hyperplasia in the lungs of animals pretreated with keratinocyte growth factor, and marked pulmonary edema in animals pretreated with alpha-naphthylthiourea. Less edema was apparent in the keratinocyte growth factor/alpha-naphthylthiourea group. All data are expressed as mean +/- SEM.

CONCLUSIONS

Pretreatment with keratinocyte growth factor significantly attenuates pulmonary edema induced by alpha-naphthylthiourea. The mechanisms of this protection are likely related to type II pneumocyte hyperplasia, but remain to be specifically elucidated.

OBJECTIVE

Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI.

METHODS

One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC).

RESULTS

Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC.

CONCLUSIONS

MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.

OBJECTIVE

The aim of the present study was to determine the dose effect of palifermin (recombinant human keratinocyte growth factor, rHuKGF) for reduction of the response of oral mucosa to fractionated radiotherapy in a mouse model.

METHODS

Ulceration (confluent mucositis) of mouse tongue epithelium was analysed as the clinically relevant endpoint. Palifermin at doses from 1 - 30 mg/kg was administered before the onset (day -3), at the end of the first (day +4) or the second week of irradiation (day +11) with 5 x 3 Gy/week. Each protocol was terminated by graded radiation test (top-up) doses. In a further experiment, optimally effective doses were given on days -3 and +4, or -3, +4 and +11.

RESULTS

Single dose irradiation of mouse mucosa yielded an ED50 (dose inducing ulcer in 50% of the mice) of 10.7 +/- 1.0 Gy. With fractionated irradiation for 1 week an ED50 for test irradiation (day +7) of 5.1 +/- 1.9 Gy was observed. After 2 weeks (day +14), the ED50 was 7.3 +/- 1.9 Gy. Palifermin significantly increased the ED50 values in all protocols tested. Maximally effective doses for single injections were 15.0 mg/kg (day -3, +11) or 22.5 mg/kg (day +4), which yielded ED50 values of 12.1 +/- 1.3 Gy, 13.7 +/- 1.5 Gy and 14.4 +/- 1.3 Gy, respectively. Higher palifermin doses did not further increase the ED50. Repeated injections on days -3 and +4 did not increase the ED50 beyond the value obtained with injections on day +4 alone. An additional injection on day +11 increased the ED50 further to 15.1 +/- 0.1 Gy.

CONCLUSIONS

A significant palifermin dose-effect was seen at doses below 15 mg/kg. However, a significant increase in oral mucosal radiation tolerance by palifermin over untreated control tissue was observed already with low doses of 1 mg/kg. This indicates that in clinical studies with palifermin, the dose of the growth factor may be of minor relevance over a wide dose range.

Hand-foot skin reaction is a most common multi-kinase inhibitor-related adverse event. This study aimed to examine whether the toxicity of sorafenib and sunitinib for human <em>keratinocytes</em> was associated with inhibiting signal transduction and activator of transcription 3 (STAT3). We studied whether STAT3 activity affects sorafenib- and sunitinib-induced cell <em>growth</em> inhibition in HaCaT cells by WST-8 assay. Stattic enhanced the cell-<em>growth</em> inhibitory and apoptotic effects of sorafenib and sunitinib. HaCaT cells transfected with constitutively-active STAT3 (STAT3C) were resistant to the sorafenib- and sunitinib-induced cell <em>growth</em> inhibition. STAT3 activity decreased after short-term treatment with sorafenib and sunitinib in a dose-dependent manner and recovered after long-term treatment with sorafenib and sunitinib at low doses. Moreover, the expression of survivin and bcl-<em>2</em> decreased after treatment with sorafenib and sunitinib was concomitant with variations in STAT3 activity. Sorafenib-induced STAT3 inhibition was mediated by regulation via MAPK pathways in HaCaT cells, while sunitinib-induced STAT3 inhibition was not. Thus, STAT3 activation mediating apoptosis suppressors may be a key <em>factor</em> in sorafenib and sunitinib-induced <em>keratinocyte</em> cytotoxicity.

BACKGROUND

The functional state of vasculature is tightly controlled by vascular endothelial <em>growth</em> <em>factor</em> receptor-<em>2</em> (VEGFR-<em>2</em>). Recent studies revealed that VEGFR-<em>2</em> is expressed on hair follicle <em>keratinocytes</em>.

OBJECTIVE

We proposed to investigate its effect on proliferation, adhesion and migration of cultured human outer root sheath cells from central hair follicle epithelium.

METHODS

These studies were undertaken in vitro using human outer root sheath cells from central hair follicle epithelium, immunohistochemistry analysis, immunofluorescence microscopy, western blot analysis, MTT, trans well analysis, and RT-PCR.

RESULTS

Our results show that VEGFR-<em>2</em> is expressed in these cells in vivo and in vitro. Furthermore, proliferation and migration of cultured human outer root sheath cells from central hair follicle epithelium is increased by VEGF165, while homotypic adhesion is decreased but heterotypic adhesion is increased. VEGF165 upregulates integrin β1 but dowregulates lgr6 expression. In addition, phosphorylation of VEGFR-<em>2</em>, Erk1/<em>2</em>, c-Jun and p38, are increased following VEGF165 treatment and these effects are reversed by a VEGFR-<em>2</em> neutralizing antibody.

CONCLUSIONS

Our results suggest a role of VEGF/VEGFR-<em>2</em> beyond angiogenesis in hair follicle regulation.

The beta and gamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma) were found to bi-directionally regulate the UV-induced activation of p38 and c-Jun NH(<em>2</em>)-terminal kinase, and the UV-induced activation of p38 was reported to enhance the resistance of normal <em>keratinocytes</em> to apoptosis. However, the signaling pathway downstream of Gbetagamma for this UV-induced p38 activation is not known. Thus, we examined the role of the Rho GTPase family in the regulation of UV-induced p38 activation by Gbetagamma. We found that overexpression of Gbetagamma increased the UV-induced activation of Cdc4<em>2</em> and that overexpression of constitutively active V1<em>2</em> Cdc4<em>2</em> increased the UV-induced p38 activation. Transfection of dominant negative N17 Cdc4<em>2</em> or small interfering RNA for Cdc4<em>2</em> blocked UV-induced p38 activation mediated by Gbetagamma in COS-1 and HaCaT cells. UV-induced p38 activation by Gbetagamma was blocked by overexpression of dominant negative p<em>2</em>1-activated kinase (PAK)-interacting exchange <em>factor</em> beta (betaPix), and wild type betaPix stimulated the UV-induced p38 activation, which was blocked by N17 Cdc4<em>2</em>. Gbetagamma increased the UV-induced activation of Ras, and the overexpression of V1<em>2</em> Ras increased UV-induced p38 activation, which was blocked by dominant negative betaPix. UV-induced p38 activation was inhibited by N17 Ras and a farnesyltransferase inhibitor, manumycin A. Gbetagamma also increased the UV-induced phosphorylation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR), and the UV-induced p38 activation was blocked by an EGFR kinase inhibitor, AG1478. From these results, we conclude that Gbetagamma mediates UV-induced activation of p38 in a Cdc4<em>2</em>-dependent way and that EGFR, Ras, and betaPix act sequentially upstream of Cdc4<em>2</em> in COS-1 and HaCaT cells.

Activation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) provides a measure of protection to immortalized epidermal <em>keratinocytes</em> (HaCaT cells) against apoptosis induced by diverse cellular stressors. This effect is due, in part, to sustained MAPK-dependent Bcl-xL expression. Here, we report a second EGFR/MAPK-dependent signaling event that protects HaCaT cells against apoptosis incurred during forced suspension culture (anoikis). This pathway targets Bim, a pro-apoptotic BH3-only Bcl-<em>2</em> family member. Bim expression was functionally relevant to HaCaT cell survival as demonstrated by partial protection against anoikis provided by siRNA-induced Bim downregulation. <em>Growth</em> <em>factor</em> starvation of attached and suspended cells was associated with enhanced Bim expression whereas EGFR activation reduced Bim expression by inducing Bim phosphorylation and proteasomal degradation. EGFR-dependent Bim phosphorylation required MAPK activation. Furthermore, PKC-delta activity contributed to both MEK/MAPK phosphorylation and Bim phosphorylation as demonstrated using both pharmacological inhibitors of PKC-delta and siRNA-mediated PKC-delta knockdown. In addition to HaCaT cells, EGFR activation supported survival and induced Bim phosphorylation in several squamous carcinoma cell lines in a strictly MAPK-dependent fashion. These results establish that EGFR activation attenuates susceptibility of immortalized and malignant <em>keratinocytes</em> to apoptosis by post-translational control of Bim-EL expression through a pathway requiring PKC-delta and MEK/MAPK activation.

OBJECTIVE

Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored.

METHODS

Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control. Retinal function was evaluated 8 weeks later by the electroretinogram and compared with photoreceptor cell layer count. LEDGF mRNA and protein levels and mRNA levels of known stress-related factors were compared in treated and control retinas to explore the mechanism of LEDGF protection. Nine RCS rats were treated with adenovirus-heat shock protein 27 (Ad-HSP27) and examined for protection.

RESULTS

Significant photoreceptor protection was evident functionally and morphologically in 65% to 100% of the RCS rats treated at early ages of up to 7 weeks. Cell protection was more prominent in the superior retinal hemisphere which has a slower natural degeneration rate in untreated eyes. Although many of the heat shock proteins and other stress-related genes showed significant elevation in the AAV-Ledgf-treated eyes, all increases were approximately twofold or less. Transduction of retinal cells with Ad-HSP27 also resulted in photoreceptor protection. AAV-Ledgf elicited no photoreceptor functional protection in P23H rhodopsin transgenic rat retina.

CONCLUSIONS

Chronic LEDGF treatment via AAV-Ledgf administration gave successful protection of photoreceptors in the RCS rat retina and retarded cell death by about 2 weeks. Induction of heat shock proteins also gave photoreceptor protection. However, compelling evidence was not found that LEDGF protection was associated with upregulation of heat shock proteins.

In this study, the effects and the mediating <em>factors</em> of dermal cells on the epidermal regenerative ability were investigated. Human epidermal cells were separated into rapidly adhering (RA) cells and slowly adhering (SA) cells and used for culturing skin equivalents (SEs). For dermal part, normal human fibroblasts, dermal sheath cells (DSCs), and dermal papilla cells were used. SEs produced using SA cells and DSCs showed a thicker epidermis and higher expressions of alpha(6)- and beta1-integrin than SEs using SA cells and normal fibroblasts showed. We hypothesized that DSCs may secrete specific cytokines that can influence the regenerative potential of epidermal cells, and compared cytokine secretion by DSCs and normal human fibroblasts. Using RayBio human cytokine antibody array C (series 1000), 1<em>2</em>0 cytokines were tested. Results showed that DSCs produced a much greater amount of insulin-like <em>growth</em> <em>factor</em>-binding protein (IGFBP-<em>2</em>), angiogenin, and BMP-6 than normal human fibroblasts produced. On the basis of the cytokine antibody array, we next investigated whether IGFBP-<em>2</em>, angiogenin, or BMP-6 has effects on SEs reconstruction. The addition of IGFBP-<em>2</em> induced a thicker and more mature epidermis and higher expressions of alpha(6)- and beta1-integrin, whereas BMP-6 exhibited little effect. Thus, the SEs with IGFBP-<em>2</em> showed almost the same morphology of the SEs using DSCs. Further, p63, a putative <em>keratinocyte</em> stem cell marker, was more frequently observed in the basal layer of SE with IGFBP-<em>2</em>. In conclusion, IGFBP-<em>2</em> is a major <em>factor</em> from DSCs that affects epidermal regenerative capacity of skin and may play an important role for stemness maintenance in human epidermal <em>keratinocytes</em>.

Non-thermal atmospheric-pressure plasmas provide new hope for improvement in chronic wound management because of their potency in killing microorganisms. However, the effectiveness of the procedure has to be verified and negative effects on healthy tissues have to be excluded. In wound healing adhesion molecules play a crucial role for cell migration and proliferation. We investigated whether an atmospheric-pressure plasma jet (kINPen09) influences the expression of adhesion molecules responsible for cell-cell and cell-matrix interactions after treatment of HaCaT-<em>keratinocytes</em> for 10 and 30 s. Twenty-four hours after plasma treatment expression of α(<em>2</em>) - and β(1)-integrin, E-cadherin and the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) was determined by flow cytometry. Plasma-treated HaCaT-cells were characterized by normal α(<em>2</em>)-integrin and increased β(1)-integrin expression. E-cadherin and EGFR expression was reduced after the 30-s treatment. We did not observe any effects following the 10-s plasma treatment. In conclusion, short-term plasma treatment can be applied without effects for cell-cell and cell-matrix adhesion.

Epidermal <em>Growth</em> <em>Factor</em> (EGF) has been identified as playing a critical role in the wound healing process. The objective of this study is to investigate the role that EGF plays in rat tympanic membrane (TM) wound healing using two techniques, microarray and immunohistochemistry. The tympanic membranes of rats were perforated using a sterile needle and sacrificed at time points during <em>2</em> weeks following perforation. The normalized signal intensities at the time points for EGF and associated genes are presented. The rat EGF mRNA did not change significantly between time points. Five associated proteins, including heparin-binding EGF-like <em>growth</em> <em>factor</em> were found to be differentially expressed above a two fold threshold at 1<em>2</em> h following perforation. EGF staining was found at low levels in the uninjured TM. Levels of EGF staining increased at <em>2</em>4 h in the basal <em>keratinocyte</em> layer, became diffusely elevated in the specimen at 36 h, before a second peak in staining of the <em>keratinocyte</em> layer at Day 4. The staining of EGF corresponds to its multiple roles in TM wound healing.

OBJECTIVE

Nuclear/radiological threats have evolved and scenarios for terrorist attacks involving radioactive material have been identified as complex situations. Mass casualty scenarios may happen, and individuals may be exposed to intentionally hidden sources of high activity, resulting in delayed diagnosis and treatment of acute radiation syndrome (ARS). Moreover, ARS must be considered as an emergency in order to better anticipate delayed radiation toxicity. In this context, therapeutic strategies in radiation casualties have to be revisited and new pharmacological approaches developed.

METHODS

B6D2F1 mice were total-body irradiated (TBI) with a 9 Gy gamma dose and then received intraperitoneal doses of either early (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin-3 [SFT3] +/- keratinocyte growth factor (KGF); stem cell factor + erythropoietin + Peg-filgrastim [SEG]) or delayed treatments (SFT3 +/- KGF, erythropoietin, or hyaluronic acid). Survival was monitored and bone marrow hematopoiesis evaluated at 300 days following early treatments.

RESULTS

SFT3 anti-apoptotic cytokine combination administered early (2 hours and 24 hours) after lethal TBI induced 60% survival versus 5% in controls. Early SEG treatment may be an alternative to SFT3 in terms of survival (55%), but SEG benefit might be obtained at the expense of long-term hematopoiesis. SFT3 + KGF induced 75% survival. No effectiveness was observed, over antimicrobial supportive care, when administration of SFT3 or its tested combinations was delayed at 48 hours.

CONCLUSIONS

As a potentially multi-organ failure, ARS requires global therapy, beyond the hematopoietic syndrome, which may include pleiotropic cytokines such as KGF.

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma <em>growth</em> but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow <em>growth</em>, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected <em>keratinocytes</em> and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal <em>growth</em> <em>factor</em> (EGF)-dependent extracellular signal-regulated kinase 1/<em>2</em> (ERK1/<em>2</em>) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of <em>keratinocytes</em>.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a fibroblast <em>growth</em> <em>factor</em> which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the <em>growth</em> <em>factor</em> and of its receptor. Western blot analysis with anti-FGFR<em>2</em> and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A<em>2</em>53 epithelial tumor cells and human cultured <em>keratinocytes</em> allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.

Epidermal regeneration is a complex process, strongly influenced by <em>growth</em> <em>factors</em>, including <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). The objective of this study was to establish immortalized HaCaT <em>keratinocytes</em> and KMST-6-fibroblasts stably expressing KGF. Transfection efficiency, genomic integration, and functionality of the transgene were determined by ELISA and PCR, and KGF-expressing clones were selected using an air-liquid interface test system. HaCaT cells displayed stronger transgene expression compared to transfected fibroblasts, and the most effective HaCaT clone was incubated on a membrane carrier to form a "membrane cell graft." Twenty-one superficial second-degree burn wounds were created in each of three pigs, and wound healing capacity of the generated "polypeptide cell delivery system" after grafting was examined. Untransfected HaCaT <em>keratinocytes</em> and membrane-covered and untreated burn wounds served as controls. Histological and macroscopical follow-up revealed that grafting of transfected HaCaT cells resulted in complete reepithelialization within 5 days, while wounds covered with untransfected cells needed <em>2</em> days longer. At untreated sites, a thin epithelium was detectable after 10 days. The results indicate that wound healing processes can be stimulated distinctly by <em>growth</em> <em>factors</em> secreted from HaCaT cells, with a prominent role for transgenic KGF.