BACKGROUND

Acute lung injury is a common, devastating clinical syndrome associated with substantial mortality and morbidity with currently no proven therapeutic interventional strategy to improve patient outcomes. The objectives of this study are to test the potential therapeutic effects of keratinocyte growth factor for patients with acute lung injury on oxygenation and biological indicators of acute inflammation, lung epithelial and endothelial function, protease:antiprotease balance, and lung extracellular matrix degradation and turnover.

METHODS

This will be a prospective, randomised, double-blind, allocation-concealed, placebo-controlled, phase 2, multicentre trial. Randomisation will be stratified by presence of severe sepsis requiring vasopressors. Patients in an ICU fulfilling the American-European Consensus Conference Definition of acute lung injury will be randomised in a 1:1 ratio to receive an intravenous bolus of either keratinocyte growth factor (palifermin, 60 μg/kg) or placebo (0.9% sodium chloride solution) daily for a maximum of 6 days. The primary endpoint of this clinical study is to evaluate the efficacy of palifermin to improve the oxygenation index at day 7 or the last available oxygenation index prior to patient discontinuation from the study.A formal statistical analysis plan has been constructed. Analyses will be carried out on an intention-to-treat basis. A single analysis is planned at the end of the trial. P = 0.05 will be considered statistically significant and all tests will be two-sided. For continuously distributed outcomes, differences between groups will be tested using independent-sample t tests, analysis of variance and analysis of covariance with transformation of variables to normality or nonparametric equivalents. The trial will be reported in line with the Consolidated Standards of Reporting Trials (Consort 2010 guidelines).

BACKGROUND

ISRCTN95690673.

Cutaneous wound repair involves extracellular matrix degradation, cell migration, matrix resynthesis and tissue remodeling. In the rodent, transcriptional regulation of collagenase-3 (MMP-13) most likely plays a role in these processes. Therefore, we isolated and characterized a 1.76-kb 5'-flanking region of the mouse MMP-13 gene. Assay of promoter activity by transient transfection of HT1080 cells and primary mouse skin fibroblasts allowed identification of several functional regions of the 5'-flanking DNA. Expression of luciferase reporter constructs in these cells was induced by phorbol myristate acetate (PMA), but not by transforming <em>growth</em> <em>factor</em>-beta(<em>2</em>) (TGF-beta(<em>2</em>)). To study the regulation of MMP-13 in cutaneous wound healing, we generated transgenic mouse lines harboring the firefly luciferase reporter gene under control of a 660-bp mouse MMP-13 promoter which showed maximal response. MMP-13 mRNA levels in transgenic lung fibroblasts increased 1.5-<em>2</em>.6-fold after PMA challenge. MMP-13 promoter activity in wounds was visualized and quantified in vivo as luciferase bioluminescence. MMP-13 expression was present at day 1 and maximal at day 18 post-wounding. Luciferase activity progressed from the wound margin towards the center of the wound. In situ hybridization showed the same spatial and temporal patterns for the luciferase and endogenous MMP-13 mRNA. Both signals localized predominantly to dermal fibroblasts at the wound periphery but not to granulation tissue or to <em>keratinocytes</em>. These results suggested that MMP-13 participated in the wound healing of acute wounds, and it was a significant <em>factor</em> in long-term remodeling of wound connective tissue in rodent skin.

Tumor necrosis <em>factor</em>-alpha (TNF-alpha) is an important proinflammatory cytokine involved in the pathogenesis of inflammatory skin diseases and cutaneous squamous cell carcinoma. Some of these effects are mediated by the stimulatory effect of this cytokine on the Akt signalling pathway, which renders <em>keratinocytes</em> less susceptible to proapoptotic stimuli and enhances cell <em>growth</em>. We have recently shown that TNF-alpha-induced Akt activation may promote the early stages of skin cancer. In this work, we demonstrate that in the premalignant <em>keratinocyte</em> cell line HaCaT, TNF-alpha activates Akt, ERK1/<em>2</em> and p38. The specific peptide blocking the activity of the atypical protein kinase C (aPKC) species zeta and iota/lambda abrogated the effects of TNF-alpha on Akt and ERK1/<em>2</em> but increased the activation of p38. The TNF-alpha-dependent phosphorylation of Akt-ERK1/<em>2</em> was slightly decreased by NF kappaB inhibition and in the presence of p38 blockers. Akt/ERK signalling but not p38 activation was abolished in the presence of the iron chelator desferroxamine that blocks formation of hydroxyl ( OH) radicals. Thus, the TNF-alpha signalling in <em>keratinocytes</em> seems to bifurcate into an aPKC-, NFkB- and OH-dependent pathway resulting in the activation of survival and mitogenic pathways mediated by Akt and ERK1/<em>2</em>, and a signalling pathway conveyed by p38 that contributes to Akt activation but is suppressed by aPKC. Our data may be utilized in the development of more selective anti-TNF-alpha therapeutic strategies.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a member of the heparin-binding fibroblast <em>growth</em> <em>factor</em> family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, though from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-<em>2</em> gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and analysis of agents regulating KGF expression reinforced the idea that KGF acts predominantly on epithelial cells. While the data do not implicate a KGF autocrine loop in neoplasia, paracrine sources of <em>factor</em> or ligand-independent signaling by the KGFR might contribute to malignancy. Alternatively, because of its differentiation-promoting effects, KGF may retard processes that culminate in uncontrolled cell <em>growth</em>.

This review presents evidence for the immunopathogenesis of psoriasis. T lymphocytes with human lymphocyte antigen (HLA)-DR molecules and receptors for interleukin <em>2</em> were found in the dermis of psoriatic plaques, suggesting the presence of activated T cells in these lesions. <em>Keratinocytes</em> in active plaques demonstrated HLA-DR molecules on their surfaces. These immunologic abnormalities were reversible with medical therapy. <em>Keratinocyte</em> HLA-DR expression was associated with an increased incidence of psoriatic arthritis. We propose that HLA-DR + <em>keratinocytes</em> and Langerhans cells in plaques could activate dermal T cells directly in an autologous mixed leucocyte/epithelial cell reaction. Alternatively, they could present an unknown autologous or exogenous antigen to T lymphocytes. T cell activation would then lead to the release of mediators of inflammation, and possibly of epidermal <em>growth</em> <em>factors</em>. This hypothesis also provides an explanation for the chronicity of psoriasis. Most of the therapies used to treat psoriasis suppress cellular immune function and inflammation. These include ultraviolet irradiation, cyclosporine, corticosteroids, methotrexate, anthralin, and retinoids.

The initiation of premalignant lesions is associated with subtle cellular and gene expression changes. Here we describe a severe combined immunodeficiency mouse xenograft model with human adult skin and compare chemical carcinogenesis and wound healing. We focus on a secreted binding protein for fibroblast <em>growth</em> <em>factors</em> (FGF-BP) that enhances the activity of locally stored FGFs and is expressed at high levels in human epithelial cancers. Carcinogen treatment of murine skin induced papilloma within 6 weeks, whereas the human skin grafts displayed no obvious macroscopic alterations. Microscopic studies of the human skin, however, showed p53-positive <em>keratinocytes</em> in the epidermis, increased angiogenesis in the dermis of the treated skin, enhanced proliferation of <em>keratinocytes</em> in the basal layer, and an increase of FGF-BP protein and mRNA expression. In contrast, after surgical wounding of human skin grafts or of mouse skin, FGF-BP expression was upregulated within a few hours and returned to control levels after <em>2</em> days with wound closure. Enhanced motility of cultured <em>keratinocytes</em> and dermal fibroblasts by FGF-BP supports a role in wound healing. We conclude that adult human skin xenografts can be used to identify early molecular events during malignant transformation as well as transient changes during wound healing.

Skin substitutes are increasingly being used in the treatment of difficult to heal wounds but their mechanisms of action are largely unknown. In this study, using histology, immunostaining, flow cytometry, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction, we determined the response to injury of a human bilayered skin substitute. Meshing or scalpel fenestration of the construct was found to stimulate <em>keratinocyte</em> migration and to decrease proliferation. By <em>2</em>4 h, flow cytometry of the <em>keratinocyte</em> component showed that meshing was associated with a 33% decrease in the number of cells in S phase (p < 0.01). An approximately <em>2</em>-fold decrease in staining for Ki67, a proliferation marker, was observed with meshing of human bilayered skin substitute. The process of reepithelialization was apparent by 1<em>2</em> h, however, the wounded human bilayered skin substitute was healed by day 3, and a stratum corneum and fully stratified epithelium were re-established by day 4. Reverse transcription polymerase chain reaction analysis and enzyme-linked immunosorbent assays showed that the expression of acute proinflammatory cytokines (interleukins 1alpha, 6, and 8, tumor necrosis <em>factor</em> alpha) peaked by 1<em>2</em>-<em>2</em>4 h postinjury. The levels of mRNA of certain <em>growth</em> <em>factors</em> (transforming <em>growth</em> <em>factor</em> beta1, vascular endothelial <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> <em>2</em>) but not others (platelet-derived <em>growth</em> <em>factors</em> A and B, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factors</em> 1 and 7, transforming <em>growth</em> <em>factor</em> beta3) increased by 1<em>2</em> h and peaked by 1-3 d after injury, returning to normal by day 6. Immunostaining for tumor necrosis <em>factor</em> alpha and transforming <em>growth</em> <em>factor</em> beta1 paralleled these findings by reverse transcription polymerase chain reaction. We conclude that human bilayered skin substitute, as a prototypic bilayered skin substitute, is a truly dynamic living tissue, capable of responding to physical injury in a staged and specific pattern of cell migration, reepithelialization, and cytokine expression.

Human fibrillin-1 is the major structural protein of extracellular matrix 10-1<em>2</em> nm microfibrils. It has a disulfide-rich modular organization which consists primarily of cbEGF (Ca(<em>2</em>+)-binding epidermal <em>growth</em> <em>factor</em>-like) domains and TB (transforming <em>growth</em> <em>factor</em> beta-binding protein-like) domains. TB4 contains an RGD (Arg-Gly-Asp) integrin-binding motif. The atomic structure of this region has been solved by X-ray crystallography and shows the TB4 and flanking cbEGF domains to be arranged as a tetragonal pyramid with N- and C-termini exposed at opposite ends of the fragment. The RGD integrin-binding motif is located within a flexible loop. We have used a variety of biophysical, biochemical and cell biology methods to investigate the molecular properties of integrin-fibrillin-1 interactions and have demonstrated that recombinant fibrillin-1 domain fragments mediate binding to integrins alphaVbeta3, alpha5beta1 and alphaVbeta6. Integrin alphaVbeta3 is a high-affinity fibrillin-1 receptor (K(d) approximately 40 nM), whereas integrins alphaVbeta6 and alpha5beta1 show moderate-affinity (K(d) approximately 450 nM) and low-affinity (K(d) >1 microM) binding respectively. Different patterns of alpha5beta1 distribution are seen when human <em>keratinocytes</em> and fibroblasts are plated on to fibrillin domain fragments compared with those seen for fibronectin, suggesting that fibrillin may cause a lesser degree or different type of intracellular signalling. A number of disease-causing mutations which affect the TB4 domain have been identified. These are being investigated for their effects on integrin binding and/or changes in intramolecular structure.

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal <em>growth</em> <em>factor</em> (EGF) receptors. Since EGF has an important role in epithelial <em>growth</em> and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (<em>2</em>- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal <em>keratinocytes</em>, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the <em>growth</em> of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.

Epidermal <em>growth</em> <em>factor</em> (EGF) and transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) stimulate <em>keratinocyte</em> migration on collagen by up-regulating the alpha <em>2</em> subunit of the collagen integrin, alpha <em>2</em> beta 1. Interleukin-1 (IL-1) is an autocrine <em>factor</em>, produced by <em>keratinocytes</em> themselves, that is modulated by ultraviolet light and increases the proliferative potential of <em>keratinocytes</em> in culture. The autocrine nature of <em>keratinocyte</em>-derived IL-1 alpha is emphasized by the fact that it induces the <em>keratinocyte</em> to synthesize IL-1 alpha and TGF-alpha, a cytokine known to induce <em>keratinocyte</em> motility. Further, topical application of IL-1 alpha has been shown to promote wound healing in animals. In this study, we used a well-defined <em>keratinocyte</em> migration assay to assess the effect of IL-1 alpha on <em>keratinocyte</em> motility and to examine whether the IL-1 alpha/TGF alpha pathway is involved. The addition of recombinant human IL-1 alpha to <em>keratinocytes</em> produced a statistically significant and concentration-dependent increase in migration on matrices of collagen types I and IV, but not on laminin. Maximal levels of <em>keratinocyte</em> migration obtained on these matrices with IL-1 alpha were comparable to those obtained with stimulation by EGF and TGF-alpha. The effects of TGF-alpha and IL-1 alpha on <em>keratinocyte</em> migration are additive; however, the maximal level of migration achieved by using IL-1 alpha and TGF-alpha in combination never exceeds the maximal level of migration found by using either cytokine alone. The time course of <em>keratinocyte</em> migration induced by IL-1 alpha is delayed (onset of migration 9-1<em>2</em> h after addition) as compared with that induced by TGF-alpha (onset of migration 6-9 h after addition) even if the cells are preincubated in IL-1 alpha. Flow cytometry analysis demonstrated no change in surface expression of integrin subunits, specifically that of integrin subunit alpha <em>2</em>, previously shown to be up-regulated by EGF/TGF-alpha. These results suggest that IL-1 alpha stimulates <em>keratinocyte</em> migration on collagen via a mechanism distinct from that of EGF/TGF-alpha.

The proteinase-activated receptor PAR(<em>2</em>) has been demonstrated to modulate tumor <em>growth</em>, invasion and metastasis in various tissues. However, the role of PAR(<em>2</em>) in cutaneous cancerogenesis is still unknown. Here we could show a protective role of PAR(<em>2</em>) in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. Tumors started to appear after eight weeks. After 13 weeks, PAR(<em>2</em>)-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild type (eight tumors per mouse). Analysis of possible signal transduction pathways activated upon PAR(<em>2</em>) stimulation in HaCaT <em>keratinocytes</em> showed an involvement of extracellular signal-regulated kinase 1/<em>2</em> and profound epidermal <em>growth</em> <em>factor</em> receptor transactivation, leading to secretion of the tumor-suppressing <em>factor</em> transforming <em>growth</em> <em>factor</em>-beta1. Thus, our results provide early experimental evidence for a tumor-protective role of PAR(<em>2</em>).

Overexpression of transforming <em>growth</em> <em>factor</em>-beta1 (TGFbeta1) in the normal epidermis can provoke an inflammatory response, but whether this occurs within a developing tumor is not clear. To test this, we used an inducible transgenic mouse to overexpress TGFbeta1 in premalignant squamous lesions. Within 48 hours of TGFbeta1 induction, there was an increase in IL-17 production by both CD4(+) and gammadelta(+) T cells, together with increased expression of T-helper-17 (Th17)-polarizing cytokines. Induction of TGFbeta1 in premalignant primary <em>keratinocytes</em> elevated the expression of proinflammatory and Th17-polarizing cytokines, and the <em>keratinocyte</em>-conditioned media caused IL-17 production by naive T cells that was dependent on T-cell TGFbeta1 signaling. Microarray analysis showed significant upregulation of proinflammatory genes <em>2</em> days after TGFbeta1 induction, and this was followed by increased MPO(+), F4/80(+), and CD8(+) cells in tumors, increased CD8(+) effectors and IFNgamma(+) cells in skin-draining LNs, and tumor regression. In parallel, the percentage of tumor CD11b(+)Ly6G(+) neutrophils was reduced. Neutralization of IL-17 blocked TGFbeta1-induced CD11b(+) Ly6G(-) tumor infiltration but did not alter the reduction of neutrophils or tumor regression. Thus, TGFbeta1 overexpression causes IL-17-dependent and IL-17-independent changes in the premalignant tumor inflammatory microenvironment.

Acute irreparable UV-induced DNA damage leads to apoptosis of epidermal <em>keratinocytes</em> (KC) and the formation of sunburn cells, whereas less severely damaged cells survive but harbor the potential of tumor formation. Here we report that hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) prevents UVB-induced apoptosis in primary KC cultured in vitro. When we analyzed the signaling pathways initiated by the HGF/SF receptor c-met, we found that the phosphatidylinositol (PI) 3-kinase and its downstream-element AKT and the mitogen-activated protein (MAP) kinase were activated. Inhibition of PI 3-kinase led to a complete abrogation of the anti-apoptotic effect of HGF/SF, whereas blockade of the MAP kinase pathway had no effect. In contrast to the observation with primary KC, HGF/SF could not enhance survival after UVB irradiation of HaCaT and A431 cell lines, despite the fact that in these cells the PI 3-kinase and MAP kinase pathways were also activated by HGF/SF. Cell cycle analysis of KC revealed a G(<em>2</em>)/M arrest after UVB irradiation and a complete loss of proliferating cells. Because HGF/SF in the skin is produced by dermal fibroblasts, our findings suggest that the HGF/SF-mediated rescue of KC from apoptosis represents an important paracrine loop by which UVB-damaged KC can be kept alive to maintain the epidermal barrier function but cannot further proliferate, thereby preventing the induction of epithelial skin tumors.

Basic fibroblast <em>growth</em> <em>factor</em> (bFGF) is a mitogen for normal human melanocytes and <em>keratinocytes</em> in culture. Experiments in vitro suggest that <em>keratinocytes</em> supply bFGF to melanocytes through a paracrine mechanism and that the aberrant expression of bFGF in melanomas confers <em>growth</em> independence from bFGF-producing cells. To determine whether bFGF is expressed in vivo, we examined a series of benign and malignant melanocytic lesions in situ using bFGF riboprobes on tissue sections, and correlated bFGF expression with histologic phenotype. Seventeen melanocytic neoplasms were studied, including four common acquired nevi, four dysplastic nevi, four primary malignant melanomas, and five metastatic melanomas. Nevic cells in benign intradermal nevi showed low signal intensity (1+), whereas compound and dysplastic nevi showed <em>2</em>+ to 3+ expression in the junctional nevic cell population and 1+ expression in the dermal nevic cell population. Melanocytes in primary melanomas had intermediate (<em>2</em>+) and those in metastatic melanomas had low (1+) levels of bFGF gene transcripts. Fibroblasts expressed high levels (3+) and epidermal and adnexal <em>keratinocytes</em> moderate (<em>2</em>+) levels of bFGF in all cases studied. Basic FGF expression in endothelial cells, known to produce and respond to this <em>growth</em> <em>factor</em> in vitro, was lower than that in the fibroblast and <em>keratinocyte</em> cell population and, in 10 of 17 cases, no bFGF mRNA was detectable. This study shows that bFGF is expressed in nevomelanocytes in vivo in all melanocytic lesions studied and thus cannot be used as a marker for transformation. The presence of bFGF gene transcripts in the various dermal cell types and in <em>keratinocytes</em> suggests that it may act as an autocrine and paracrine <em>growth</em> <em>factor</em> in regulating cellular proliferation in the skin.

The hypothesis that incorporation of small amounts (0.3% w/w) of modified heparin in thiol-modified hyaluronan or HA and gelatin hydrogels would regulate release of cytokine <em>growth</em> <em>factors</em> (GFs) from those gels has been investigated in vitro. In addition, the physiologic response to gel implantation has been evaluated in vivo. Tests were performed with 6 GFs: basic fibroblast <em>growth</em> <em>factor</em> (bFGF), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), angiopoietin-1 (Ang-1), <em>keratinocyte</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-AA (PDGF), and transforming <em>growth</em> <em>factor</em>-beta 1. Release profiles for all 6 over several weeks were well fit by first order exponential kinetics (R(<em>2</em>)>> 0.9 for all cases). The most remarkable result of the experiment was a dramatic variation in the total mass ultimately released, which varied from as much as 90.<em>2</em>% of the initial load for bFGF to as little as 1.8% for PDGF, a 45-fold difference. Furthermore, gels containing either VEGF of Ang-1 produced twice the vascularization response in vivo as gels not containing a <em>growth</em> <em>factor</em>. Thus, those GFs maintained strong physiologic effectiveness.

The proliferation of normal human fibroblasts, <em>keratinocytes</em>, and melanocytes in vitro can be controlled by purified polypeptide <em>growth</em> <em>factors</em> and serum. We have studied the cellular expression of the heparin-binding <em>growth</em> <em>factor</em> type <em>2</em>/basic fibroblast <em>growth</em> <em>factor</em> (HBGF-<em>2</em>/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, <em>2</em>.<em>2</em>, and 1.5 kilobases, which hybridize to a specific HBGF-<em>2</em>/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified <em>growth</em> <em>factors</em> tested, transforming <em>growth</em> <em>factor</em> type-beta also increased HBGF-<em>2</em>/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF-<em>2</em>/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly <em>growing</em> in serum-free medium and at higher levels in cells rapidly <em>growing</em> in serum-containing medium. In contrast to fibroblasts, mRNA coding for HBGF-<em>2</em>/bFGF is undetectable in proliferating normal human <em>keratinocytes</em>, melanocytes, or mammary epithelial cells. Because <em>keratinocytes</em> and melanocytes proliferate in response to purified HBGF-<em>2</em>/bFGF, our results suggest that HBGF-<em>2</em>/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC-<em>2</em>5) expresses mRNA coding for HBGF-<em>2</em>/bFGF, suggesting that the gene may become activated in some carcinomas.

UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, including MAPKs. The betagamma subunit of the heterotrimeric GTP-binding protein (Gbetagamma) was found to mediate UV-induced p38 activation via epidermal <em>growth</em> <em>factor</em> receptor (EGFR). However, it is not known how Gbetagamma mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human <em>keratinocytes</em> with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal <em>keratinocytes</em> and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of Gbeta(1)gamma(<em>2</em>) increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of Gbetagamma by the carboxyl terminus of G protein-coupled receptor kinase <em>2</em> (GRK<em>2</em>ct) produced the opposite effect. The activation of Src increased UVB-induced, Gbetagamma-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of Gbetagamma increased UVB-induced apoptosis, and the overexpression of GRK<em>2</em>ct decreased this apoptosis. We conclude that Gbetagamma mediates UVB-induced human <em>keratinocyte</em> apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn(-/-)) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in <em>growth</em> <em>factor</em> binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn(-/-) and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn(-/-) skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS<em>2</em>,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl <em>2</em>-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn(-/-) CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast <em>growth</em> <em>factor</em> (Fgf)-7 and -<em>2</em> binding due to changes in the micro-heterogeneity of skin Dcn(-/-) CS/DS. To better delineate the role of decorin, we used a 3D Dcn(-/-) fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn(-/-) fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl <em>2</em>-O sulfotransferase in the Dcn(-/-) fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn(-/-) samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to <em>keratinocytes</em> in a concentration dependent manner unlike the Dcn(-/-) CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, <em>keratinocyte</em> proliferation was differentially affected. Higher concentration of Dcn(-/-) CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and <em>keratinocytes</em> showed that, unlike Dcn(-/-) CS/DS, wild-type CS/DS promoted differentiation of <em>keratinocytes</em>. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn(-/-) mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence <em>keratinocyte</em> behavior.

The introduction of the techniques of molecular biology as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer pathogenesis: initiation, exogenous promotion, premalignant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozygous activating mutations in c-rasHa are dominant, and affected <em>keratinocytes</em> hyperproliferate and are resistant to signals for terminal differentiation. An important pathway impacted by c-rasHa activation is the protein kinase C (PKC) pathway, a major regulator of <em>keratinocyte</em> differentiation. Increased activity of PKC alpha and suppression of PKC delta by tyrosine phosphorylation contribute to the phenotypic consequences of rasHa gene activation in <em>keratinocytes</em>. Tumor promoters disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differentiation and enhanced <em>growth</em> rate of initiated cells impart a <em>growth</em> advantage when the epidermis is exposed to promoters. The frequency of premalignant progression varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the alpha 6 beta 4 integrin and are deficient in transforming <em>growth</em> <em>factor</em> beta 1 and beta <em>2</em> peptides, two changes associated with a very high proliferation rate in this subset of tumors. The introduction of an oncogenic rasHa gene into epidermal cells derived from transgenic mice with a null mutation in the TGF beta 1 gene have an accelerated rate of malignant progression when examined in vivo. Thus members of the TGF beta gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasHa transduced <em>keratinocytes</em> from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant conversion by initiated <em>keratinocytes</em> from TG beta 1 and p53 null geneotypes suggest that a common, <em>growth</em>-related pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis model.

We investigated <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H<em>2</em>(k)) recipient mice were transplanted with C57BL/6 (H<em>2</em>(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.<em>2</em> and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th<em>2</em> cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and <em>2</em>) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th<em>2</em> cytokines in situ.

Interleukin-<em>2</em><em>2</em> (IL-<em>2</em><em>2</em>) is one of the key mediators of <em>keratinocyte</em> alterations in psoriasis. IL-<em>2</em><em>2</em> inhibits <em>keratinocyte</em> differentiation and induces the migration of human <em>keratinocytes</em>. Grb<em>2</em>-associated binder 1 (Gab1) has been shown to mediate epidermal <em>growth</em> <em>factor</em>-induced epidermal <em>growth</em> and differentiation via interaction with the Src homology-<em>2</em>-containing protein-tyrosine phosphatase (Shp<em>2</em>). In this investigation, we explore the role of Gab1 and Gab<em>2</em> in IL-<em>2</em><em>2</em>-mediated <em>keratinocyte</em> activities. We show that both Gab1 and Gab<em>2</em> were tyrosine phosphorylated in IL-<em>2</em><em>2</em>-stimulated HaCaT cells and human primary epidermal <em>keratinocytes</em> and contributed to the activation of Extracellular signal regulated kinase 1/<em>2</em> (Erk1/<em>2</em>) through interaction with Shp<em>2</em>. We further demonstrate that HaCaT cells infected with adenoviruses expressing Shp<em>2</em>-binding-defective Gab1/<em>2</em> mutants exhibited decreased cell proliferation and migration, as well as increased differentiation. Moreover, similar results were observed in HaCaT cells infected with adenovirus-based small interfering RNAs targeting Gab1 and/or Gab<em>2</em>. Altogether, these data underscore the critical roles of Gab1 and Gab<em>2</em> in IL-<em>2</em><em>2</em>-mediated HaCaT cell proliferation, migration, and differentiation.

Neu differentiation <em>factor</em> (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-<em>2</em>/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal <em>growth</em> <em>factor</em> (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-<em>2</em>/neu, and HER-3 are present in skin, and other EGF family members can influence wound <em>keratinocytes</em> in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha <em>2</em> (rhNDF-alpha <em>2</em>), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm<em>2</em>. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta <em>2</em> had no apparent biologic effects in this model. rhNDF-alpha <em>2</em> also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha <em>2</em>-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-<em>2</em>/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-<em>2</em>/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha <em>2</em> may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.

<em>Growth</em> <em>factors</em> are postulated to mediate stromal-epithelial interactions in the prostate to maintain normal tissue physiology. Transforming <em>growth</em> <em>factor</em>-beta (TGFbeta) has been shown to influence the prostate and probably mediate stromal-epithelial interactions. TGFbeta1 messenger RNA (mRNA) expression is stimulated after castration and can be suppressed by in vivo treatment with androgens. The hypothesis tested is that TGFbeta is regulated not only by androgen, but also by a network of locally produced <em>growth</em> <em>factors</em> that influence prostatic <em>growth</em> and differentiation. Epithelial and stromal cells from <em>2</em>0-day-old rat ventral prostate were isolated and used to test this hypothesis. The expression of mRNA for TGFbeta1, -<em>2</em>, and -3 was analyzed by a quantitative RT-PCR procedure. Observations from this assay demonstrate that both epithelial and stromal cells express the mRNA for TGFbeta1, -<em>2</em>, and -3. TGFbeta1 mRNA expression was constant during development of the prostate. TGFbeta<em>2</em> mRNA expression was elevated at birth, then declined and elevated again at 100 days of age. TGFbeta3 mRNA expression was high during puberty and young adult ages then declined at 100 days of age. TGFbeta<em>2</em> and TGFbeta3 expression are inversely related during prostate development. After castration of 60-day-old rats, both TGFbeta1 and TGFbeta<em>2</em> mRNA were enhanced. Interestingly, TGFbeta3 mRNA was significantly suppressed after castration. Epidermal <em>growth</em> <em>factor</em> (EGF) stimulated TGFbeta1 mRNA expression in stromal cells (6-fold increase), whereas <em>keratinocyte</em> <em>growth</em> <em>factor</em> stimulated TGFbeta<em>2</em> mRNA in epithelial cells. TGFbeta inhibited both testosterone- and EGF-stimulated prostatic stromal and epithelial cell <em>growth</em>. EGF and TGFbeta also inhibited prostatic ductal morphogenesis and <em>growth</em> in organ culture. Immunocytochemical localization of TGFbeta in <em>2</em>0-day-old prostate demonstrated predominately stromal localization of the protein. These results indicate that the isoforms of TGFbeta<em>2</em> and TGFbeta3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFbeta expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFbetas are regulated indirectly through a complex network of <em>growth</em> <em>factors</em>, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and <em>growth</em> in organ culture is postulated to be in part mediated by the increase in TGFbeta1 expression. In summary, a network of <em>growth</em> <em>factor</em>-mediated stromal-epithelial interactions is needed to maintain prostate <em>growth</em> and development. TGFbeta is postulated to have an important role in this process.

Keratin 14 (K14) is believed to play a pivotal role in the maintenance of epidermal cell shape and contributing to their resistance to mechanical trauma, thereby protecting the cells from lysing. Mice harboring a K14 null mutation produce phenotypic characteristics of epidermolysis bullosa simplex, a skin blistering disease (Lloyd et al., 1995, J Cell Biol 1<em>2</em>9:13<em>2</em>9-1344). K14 null animals die several days after birth, making the detailed study of the consequences of K14 deletion in epidermal cell physiology in vivo particularly difficult. To define the consequences of K14 loss more precisely, we used an in vitro approach by isolating K14-/- cell lines and studying epidermal differentiation in the K14 null background. Several <em>keratinocyte</em> cell lines were generated from 6-day-old mice homozygous for a targeted disruption of the K14 gene (lines designated MKC-5, MKC-<em>2</em>3, and MKC-33) and from their wild-type littermates (lines designated MKC-1 and MKC-6). Under low Ca<em>2</em>+ (0.066 mM) and low serum (<em>2</em>%) conditions, both wild-type and mutant cells were able to adhere to collagen type I-coated dishes and form epithelial sheets. They maintained basal epidermal cell characteristics and continued to proliferate without obvious signs of terminal differentiation; however, K14-/- cells proliferated two- to threefold slower than did their wild-type counterparts. The distribution of K5, the natural partner of K14, at the immunofluorescence level was also normal looking in the K14-/- MKC-5 cells, but with fewer filaments detectable, consistent with the approximately <em>2</em>0% reduction in K5 detectable on immunoblots. K17 expression was increased approximately 40% in the K14-/- cells. The levels of K15 and K16 were not different in the MKC-5 and MKC-6 cell lines, suggesting that they are not contributing <em>factors</em> to the stabilization of K5 in the mutant cells. K8, K19, and vimentin were undetectable in both lines. Both MKC-5 and MKC-6 cells underwent morphological and biochemical differentiation in response to a switch to high Ca<em>2</em>+ medium. These findings indicate that K14-/- MKC-5 cells preserve the morphological, biochemical, and physiological characteristics of epidermal cells for an extensive period of time in vitro, likely due to the compensatory expression of K17. The culturing capacity of these cells also permits the analysis of <em>keratinocyte</em> <em>growth</em> and differentiation in the absence of K14. In addition, the culturing methods we describe will be useful for the generation of epithelial cell lines from a wealth of increasingly available knockout mouse strains with early lethality.