Acute lung injury and the acute respiratory distress syndrome are common syndromes with a high mortality rate that affect both medical and surgical patients. Better understanding of the pathophysiology of acute lung injury and the acute respiratory distress syndrome and advances in supportive care and mechanical ventilation have led to improved clinical outcomes since the syndrome was first described in 1967. Although several promising pharmacological therapies, including surfactant, nitric oxide, glucocorticoids and lysofylline, have been studied in patients with acute lung injury and the acute respiratory distress syndrome, none of these pharmacological treatments reduced mortality. This article provides an overview of pharmacological therapies of acute lung injury and the acute respiratory distress syndrome tested in clinical trials and current recommendations for their use as well as a discussion of potential future pharmacological therapies including beta(<em>2</em>)-adrenergic agonist therapy, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, and activated protein C.

The systemic administration of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) enhances T-cell lymphopoiesis in normal mice and mice that received a bone marrow transplant. KGF exerts protection to thymic stromal cells from cytoablative conditioning and graft-versus-host disease-induced injury. However, little is known regarding KGF's molecular and cellular mechanisms of action on thymic stromal cells. Here, we report that KGF induces in vivo a transient expansion of both mature and immature thymic epithelial cells (TECs) and promotes the differentiation of the latter type of cells. The increased TEC numbers return within <em>2</em> weeks to normal values and the microenvironment displays a normal architectural organization. Stromal changes initiate an expansion of immature thymocytes and permit regular T-cell development at an increased rate and for an extended period of time. KGF signaling in TECs activates both the p53 and NF-kappaB pathways and results in the transcription of several target genes necessary for TEC function and T-cell development, including bone morphogenetic protein <em>2</em> (BMP<em>2</em>), BMP4, Wnt5b, and Wnt10b. Signaling via the canonical BMP pathway is critical for the KGF effects. Taken together, these data provide new insights into the mechanism(s) of action of exogenous KGF on TEC function and thymopoiesis.

Fibroblast <em>growth</em> <em>factors</em> (FGF) regulate the <em>growth</em> and differentiation of cells through complex combinatorial signaling pathways. There are nine ligands that interact with a family of four tyrosine kinase FGF receptors (FGFR). Diversity in FGF signaling is determined in part by the affinity of specific ligand-receptor pairs. Alternative splicing in the FGFR ligand binding domain generates additional receptor isoforms with novel ligand affinities. For example, splicing events in the ligand binding domain of FGFR<em>2</em> dramatically increases its affinity for <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF/FGF-7). We have identified an alternatively spliced form of the FGFR3 mRNA, corresponding to known splice variants of FGFRs 1 and <em>2</em>. We demonstrate both by binding studies on genetically engineered soluble receptors and by the mitogenic response of <em>growth</em> <em>factor</em>-dependent cell lines that this splice variant of FGFR3 (FGFR3 IIIb), by binding only acidic FGF (aFGF/FGF-1), has the most restricted ligand binding properties of any FGFR thus far described. Furthermore, by constructing a chimeric receptor that contains the homologous exon from FGFR<em>2</em>, we demonstrate that this single domain from FGFR<em>2</em> is sufficient to confer upon FGFR3 the ability to bind KGF/FGF-7. The uniquely limited repertoire of ligands that interact with this receptor suggests that a novel ligand for FGFR3 IIIb exists.

CONCLUSIONS

This review highlights the critical role of transforming growth factor beta (TGF-β)1-3 within different phases of wound healing, in particular, late-stage wound healing. It is also very important to identify the TGF-β1-controlling factors involved in slowing down the healing process upon wound epithelialization.

UNASSIGNED

TGF-β1, as a growth factor, is a known proponent of dermal fibrosis. Several strategies to modulate or regulate TGF's actions have been thoroughly investigated in an effort to create successful therapies. This study reviews current discourse regarding the many roles of TGF-β1 in wound healing by modulating infiltrated immune cells and the extracellular matrix.

RESULTS

It is well established that TGF-β1 functions as a wound-healing promoting factor, and thereby if in excess it may lead to overhealing outcomes, such as hypertrophic scarring and keloid. Thus, the regulation of TGF-β1 in the later stages of the healing process remains as critical issue of which to better understand.

CONCLUSIONS

One hypothesis is that cell communication is the key to regulate later stages of wound healing. To elucidate the role of keratinocyte/fibroblast cross talk in controlling the later stages of wound healing we need to: (1) identify those keratinocyte-released factors which would function as wound-healing stop signals, (2) evaluate the functionality of these factors in controlling the outcome of the healing process, and (3) formulate topical vehicles for these antifibrogenic factors to improve or even prevent the development of hypertrophic scarring and keloids as a result of deep trauma, burn injuries, and any type of surgical incision.

Acne is a chronic inflammatory disease of the pilosebaceous follicle. One of the main pathogenetic <em>factors</em> in acne is the increased proliferation of Propionibacterium acnes (P. acnes) in the pilosebaceous unit. We investigated whether direct interaction of P. acnes with <em>keratinocytes</em> might be involved in the inflammation and ductal hypercornification in acne. The capacities of different P. acnes strains to activate the innate immune response and the <em>growth</em> of cultured <em>keratinocytes</em> were investigated. We have found that two clinical isolates of P. acnes significantly induced human beta-defensin-<em>2</em> (hBD<em>2</em>) messenger RNA (mRNA) expression; in contrast a third clinical isolate and the reference strain (ATCC118<em>2</em>8) had no effect on hBD<em>2</em> mRNA expression. In contrast, all four strains significantly induced the interleukin-8 (IL-8) mRNA expression. The P. acnes-induced increase in hBD<em>2</em> and IL-8 gene expression could be inhibited by anti-Toll-like receptor <em>2</em> (TLR<em>2</em>) and anti-TLR4 neutralizing antibodies, suggesting that P. acnes-induced secretion of soluble <em>factors</em> in <em>keratinocytes</em> are both TLR<em>2</em> and TLR4 dependent. In addition, the clinical isolate P. acnes (889) was capable of inducing <em>keratinocyte</em> cell <em>growth</em> in vitro. Our findings suggest that P. acnes modulates the antimicrobial peptide and chemokine expression of <em>keratinocytes</em> and thereby contributes to the recruitment of inflammatory cells to the sites of infections.

OBJECTIVE

Various peptide growth factors have been found to exert functional effects among epithelial cell populations. This study assessed the role of certain fibroblast growth factors (FGFs) (acidic FGF, basic FGF, and keratinocyte growth factor) in the regulation of intestinal epithelial cell proliferation and restitution.

METHODS

Recombinant growth factors were added to subconfluent cultures of IEC-6, Caco-2, and HT-29 cell lines with subsequent assessment of [3H]-thymidine incorporation. The effects on an in vitro model of restitution were assessed by quantitation of cells migrating into standard wounds established in confluent monolayers of IEC-6 cells. Transforming growth factor beta (TGF-beta) content of growth factor-treated wounded monolayers was assessed by Northern blot and bioassay.

RESULTS

Acidic FGF, basic FGF, and keratinocyte growth factor caused a modest increase in proliferation of IEC-6, Caco-2, and HT-29 cell lines. Acidic FGF and basic FGF promoted intestinal epithelial cell restitution in vitro up to 10-fold, in conjunction with the enhanced expression of TGF-beta messenger RNA and protein. Promotion of IEC-6 restitution by acidic and basic FGF could be blocked by addition of immunoneutralizing anti-TGF-beta antisera.

CONCLUSIONS

FGFs that exert effects on fibroblast cells also exert effects on intestinal epithelial cell populations and may help promote epithelial cell restitution, the initial step of intestinal wound healing through a TGF-beta-dependent pathway.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and <em>growth</em>-promoting attributes primarily by acting as a coreceptor for basic fibroblast <em>growth</em> <em>factor</em> (FGF-<em>2</em>). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. <em>Growth</em> of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic <em>keratinocyte</em> <em>growth</em> <em>factor</em> (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor <em>growth</em> and neovascularization. Thus, perlecan is a potent inducer of tumor <em>growth</em> and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Thymus-dependent reconstitution of the peripheral T-cell compartment is critical for the successful outcome of bone marrow transplantation. However, graft-versus-host disease (GVHD) affects thymic stromal function and thus prevents normal T-cell maturation and selection. To determine whether cytoprotection of thymic epithelial cells (TECs) by <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) averts GVHD-related injury to the thymus, a nonirradiated murine parent->>F(1) transplantation model was investigated. Administration of KGF between days -3 and +3 of GVHD induction preserved normal thymic size, cellularity, and thymocyte phenotype when measured <em>2</em> weeks after transplantation and compared with saline-treated parent->>F(1) mice that received allogeneic transplants. Moreover, the characteristic GVHD-induced impairment in cell cycle progression of pro- and pre-T cells was prevented by KGF. However, the normal phenotypic and functional status of the thymus did not correlate with the higher number of GVHD-inducing mature donor T cells in thymi of KGF-treated mice. Importantly, extensive analysis of the different TEC populations within the thymic cortex and medulla revealed an almost normal stromal architecture and composition in GVHD mice treated with KGF. These observations are likely to reflect an indirect effect of KGF on thymopoiesis as KGF-receptor expression was demonstrated to be restricted to TECs. Thus, pharmacologic doses of KGF appear to exert a potent effect on TEC function, which in turn allows for normal T lymphopoiesis to occur during acute GVHD.

Epithelin 1 and <em>2</em> were originally purified from rat kidneys based on their ability to inhibit the <em>growth</em> of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V.L., Byles, C., Todaro, G.J., and Plowman, G.D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 791<em>2</em>-7916). This study presents the complete amino acid sequence of these two <em>growth</em> <em>factors</em> and the cloning of their cDNA from rat, mouse, and human sources. Epithelins 1 and <em>2</em> are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 1<em>2</em> cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/<em>2</em> copies of this novel 1<em>2</em>-cysteine motif, <em>2</em> of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent <em>keratinocytes</em> and fibroblasts. In contrast, epithelin <em>2</em> had no activity on these cells, but at high concentrations was capable of antagonizing the <em>growth</em> proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these <em>factors</em> as natural mediators of epithelial homeostasis.

Melanocytes, after cell division, separate and migrate along the basement membrane; they extend their dendrites and establish multiple contacts with <em>keratinocytes</em>. Once adhesion is established, <em>keratinocytes</em> control melanocyte <em>growth</em> and expression of cell surface receptors. Most melanomas arise within the epidermis (melanoma in situ) and then invade across the basement membrane. These melanoma cells escape from control by <em>keratinocytes</em> through five major mechanisms: (1) downregulation of receptors important for communication with <em>keratinocytes</em> such as E-cadherin, P-cadherin, and desmoglein, which is achieved through <em>growth</em> <em>factors</em> such as hepatocyte <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, and endothelin-1 produced by fibroblasts or <em>keratinocytes</em>; (<em>2</em>) upregulation of receptors and signaling molecules important for melanoma cell-melanoma cell and melanoma cell-fibroblast interactions such as N-cadherin, Mel-CAM, and zonula occludens protein-1; (3) deregulation of morphogens such as Notch receptors and their ligands; (4) loss of anchorage to the basement membrane because of an altered expression of cell-matrix adhesion molecules; (5) increased elaboration of metal-loproteinases. Thus, investigating normal melanocyte homeostasis helps us to better define how melanoma cells escape the microenvironment created by epidermal <em>keratinocytes</em> and how they develop new cellular partners in fibroblasts and endothelial cells, which support their <em>growth</em> and invasion.

We have isolated cDNA clones encoding the entire 170-kDa chain of epiligrin (alpha 3Ep) and a genomic clone encoding the alpha 3Ep gene (LamA3). Analysis of multiple cDNA clones revealed two distinct transcripts (alpha 3EpA and alpha 3EpB). Sequencing of the alpha 3EpA transcript indicated sequence and structural homology to laminin alpha 1 and alpha <em>2</em> chains that extend from domain IIIa through the carboxyl-terminal G domain. The alpha 3EpB transcript encodes a larger amino-terminal domain and contains additional epidermal <em>growth</em> <em>factor</em> repeats and sequences corresponding to domain IV of alpha 1 laminin. Fluorescence in situ hybridization indicated that the LamA3 gene is located on chromosome 18q11.<em>2</em>, a locus distinct from the LamA1 gene (18p11.3). The G domain of the epiligrin alpha 3 chain contains five subdomains that are individually related to the G subdomains reported for Drosophila and vertebrate laminin alpha chains. Sequence divergence within the G domain of alpha 3 epiligrin suggests that it is functionally distinct from laminin, consistent with our previous report showing that epiligrin interacts with different integrin adhesion receptors. Analysis of RNA from human foreskin <em>keratinocytes</em> (HFKs) identified multiple epiligrin transcripts that were down-regulated by viral transformation and differentiation. In contrast, epiligrin expression was up-regulated in wound sites of human skin.

With the goal of identifying genes with a differential pattern of expression between invasive cervical carcinomas (CVX) and normal cervical <em>keratinocytes</em> (NCK), we used oligonucleotide microarrays to interrogate the expression of 14,500 known genes in 11 primary HPV16 and HPV18-infected stage IB-IIA cervical cancers and four primary normal cervical <em>keratinocyte</em> cultures. Hierarchical cluster analysis of gene expression data identified <em>2</em>40 and <em>2</em>65 genes that exhibited greater than twofold up-regulation and down-regulation, respectively, in primary CVX when compared to NCK. Cyclin-dependent kinase inhibitor <em>2</em>A (CDKN<em>2</em>A/p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homolog (avian)-like<em>2</em> (v-Myb), minichromosome maintenance proteins <em>2</em>, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II alpha (TOP<em>2</em>A), ubiquitin-conjugating enzyme E<em>2</em>C, CD97 antigen, E<em>2</em>F transcription <em>factor</em> 1, and dUTP pyrophosphatase were among the most highly overexpressed genes in CVX when compared to NCK. Down-regulated genes in CVX included transforming <em>growth</em> <em>factor</em> beta 1, transforming <em>growth</em> <em>factor</em> alpha, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue <em>factor</em> pathway inhibitor-<em>2</em> (TFPI-<em>2</em>). Differential expression of some of these genes including CDKN<em>2</em>A/p16, v-Myb, PTGES, and TOP<em>2</em>A was validated by quantitative real-time PCR. Flow cytometry on primary CVX and NCK and immunohistochemical staining of formalin fixed paraffin-embedded tumor specimens from which primary CVX cultures were derived as well as from a separate set of invasive cervical cancers confirmed differential expression of the CDKN<em>2</em>A/p16 and PTGES markers on CVX versus NCK. These results identify several genes that are coordinately disregulated in cervical cancer, likely representing common signaling pathways triggered by HPV transformation. Moreover, these data obtained with highly purified primary tumor cultures highlight novel molecular features of human cervical cancer and provide a foundation for the development of new type-specific diagnostic and therapeutic strategies for this disease.

The effects of <em>growth</em> <em>factors</em>, hormones, and calcium on the <em>growth</em> and differentiation of secondary cultures of normal human pro<em>keratinocytes</em>, i.e., proliferative <em>keratinocytes</em>, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal <em>growth</em> was achieved when MCDB 153 was supplemented with either epidermal <em>growth</em> <em>factor</em> (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal <em>growth</em>. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal <em>growth</em> occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (<em>2</em> mM). This suggests that an inverse relationship exists between the <em>growth</em> response (extent of clonal <em>growth</em>) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially <em>growing</em> secondary cultures of adult and neonatal pro<em>keratinocytes</em> have a <em>2</em>4-hour cell generation time with G1, S, G<em>2</em>, and M phases of 1<em>2</em>, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be <em>growth</em> arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal pro<em>keratinocytes</em> (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

The expression of the activated mitogen-activated kinases/extracellular signal-regulated kinases (ERKs) ERK1 and ERK<em>2</em> was characterized in 101 humanhead and neck squamous carcinoma specimens. Activated ERK1/<em>2</em>were detected at different levels in the majority of these tumors, as assayed by immunostaining with an antibody specific for the dually phosphorylated and activated ERK1 and ERK<em>2</em>. ERK1/<em>2</em> activation levels were higher in tumors with advanced regional lymph node metastasis (P = 0.048) and in relapsed tumors (P = 0.0<em>2</em>1). The expression of epidermal <em>growth</em> <em>factor</em> (EGF) receptor (P = 0.037), transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha; P < 0.001), and HER<em>2</em> (P = 0.066; positive trend) correlated with activation of ERK1/<em>2</em>. In a multivariate analysis, both TGF-alpha (P < 0.0001) and HER<em>2</em> (P = 0.045) were independently correlated with ERK1/<em>2</em> activation. In turn, activation of ERK1/<em>2</em> was associated with a higher Ki-67 proliferative index (P = 0.00<em>2</em>). In EGF receptor-dependent model cells (A431 and DiFi), a specific EGF receptor tyrosine kinase inhibitor ("Iressa"; ZD1839) and a chimeric anti-EGF receptor antibody ("Cetuximab"; C<em>2</em><em>2</em>5) inhibited ERK 1/<em>2</em> activation at concentrations that inhibited autocrine cell proliferation. In patients on treatment with C<em>2</em><em>2</em>5, the activation of ERK1/<em>2</em> in skin, an EGF receptor-dependent tissue, was lower compared with control skin. Parallel changes were seen in <em>keratinocyte</em> Ki67 proliferation indexes in skin from C<em>2</em><em>2</em>5-treated patients. Taken together, these studies provide support for a role of activation of ERK1/<em>2</em> in head and neck squamous carcinoma and a correlation with EGF receptor/TGF-alpha expression. The inhibition of ERK1/<em>2</em> activation in vitro and in vivo by compounds targeting the EGF receptor points to the interest of ERK1/<em>2</em> as potential surrogate markers of EGF-receptor signaling in clinical therapeutic studies.

In order to determine whether <em>keratinocytes</em> play a role in the modulation of the immune response, we investigated the murine <em>keratinocyte</em> cell line Pam <em>2</em>1<em>2</em>. In culture these cells generate a substance with a biologic activity that greatly enhances phytohemagglutinin-induced thymocyte proliferation. We have, therefore, called this substance epidermal cell thymocyte-activating <em>factor</em> (ETAF). This <em>keratinocyte</em>-derived supernatant activity is mainly produced at the onset of the logarithmic <em>growth</em> phase and is directly mitogenic for murine thymocytes. Although ETAF by itself exhibits no T cell <em>growth</em> <em>factor</em> activity, ETAF enhances Interleukin <em>2</em> production by mitogen-stimulated murine spleen cells. Murine ETAF is not genetically restricted and lacks species specificity since it decreases lectin-induced proliferation of human peripheral blood lymphocytes (as well as murine spleen cells) and also enhances the production of human Interleukin <em>2</em>. The <em>factor</em> has a m.w. between 15,000 and <em>2</em>5,000 as determined by gel filtration and elutes as a single peak from anion exchange chromatography columns. The activity is maintained mainly at alkaline pH and is rapidly destroyed at temperatures above 60 degrees C. These observations suggest that epidermal cells may interact with the immune system by elaborating nonspecific <em>factors</em> that modulate lymphocyte proliferation and augment lymphokine production.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription <em>factors</em> that belong to the nuclear hormone receptor superfamily. PPARalpha is mainly expressed in the liver, where it activates fatty acid catabolism. PPARalpha activators have been used to treat dyslipidemia, causing a reduction in plasma triglyceride and elevation of high-density lipoprotein cholesterol. PPARdelta is expressed ubiquitously and is implicated in fatty acid oxidation and <em>keratinocyte</em> differentiation. PPARdelta activators have been proposed for the treatment of metabolic disease. PPARgamma<em>2</em> is expressed exclusively in adipose tissue and plays a pivotal role in adipocyte differentiation. PPARgamma is involved in glucose metabolism through the improvement of insulin sensitivity and represents a potential therapeutic target of type <em>2</em> diabetes. Thus PPARs are molecular targets for the development of drugs treating metabolic syndrome. However, PPARs also play a role in the regulation of cancer cell <em>growth</em>. Here, we review the function of PPARs in tumor <em>growth</em>.

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis <em>factor</em> (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis <em>factor</em> binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 1<em>2</em> antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating <em>factor</em>, and granulocyte macrophage colony stimulating <em>factor</em>; anti-interleukin <em>2</em> receptor antibody; epidermal <em>growth</em> <em>factor</em>; <em>keratinocyte</em> <em>growth</em> <em>factor</em> <em>2</em> (repifermin); human <em>growth</em> hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis <em>factor</em> receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.

Selenium (Se) is an essential trace element for animals and humans that is obtained from dietary sources including cereals, grains and vegetables. The Se content of plants varies considerably according to its concentration in soil. Plants convert Se mainly into Se-methionine (Se-Met) and incorporate it into protein in place of methionine (Met). Selenocystine (Se-Cys), methyl-Se-Cys and gamma-glutamyl-Se-methyl-Cys are not significantly incorporated into plant protein and are at relatively low levels irrespective of soil Se content. Higher animals are unable to synthesize Se-Met and only Se-Cys was detected in rats supplemented with Se as selenite. Renal regulation is the mode by which whole body Se is controlled. Se is concentrated in hair and nail and it occurs almost exclusively in organic compounds. The potentiating effect of Se deficiency on lipid peroxidation is enhanced in some tissues by concurrent deficiency of copper or manganese. In the in vitro system, the chemical form of Se is an important <em>factor</em> in eliciting cellular responses. Although the cytotoxic mechanisms of selenite and other redoxing Se compounds are still unclear, it has been suggested that they derive from their ability to catalyze the oxidation of thiols and to produce superoxide simultaneously. Selenite-induced cytotoxicity and apoptosis in human carcinoma cells can be inhibited with copper (CuSO(4)) as an antioxidant. High doses of selenite result in induction of 8-hydroxydeoxyguanosine (8-OHdG) in mouse skin cell DNA and in primary human <em>keratinocytes</em>. It may cause DNA fragmentation and decreased DNA synthesis, cell <em>growth</em> inhibition, DNA synthesis, blockade of the cell cycle at the S/G(<em>2</em>)-M phase and cell death by necrosis. In contrast, in cells treated with methylselenocyanate or Se methylselenocysteine, the cell cycle progression was blocked at the G(1) phase and cell death was predominantly induced by apoptosis.

Since heparin-binding epidermal <em>growth</em> <em>factor</em> (HB-EGF) is a member of the EGF family and binds to EGF receptor, we tested recombinant HB-EGF for its ability to stimulate human <em>keratinocyte</em> proliferation. The effect of HB-EGF on human <em>keratinocytes</em> was dependent on the cell density. HB-EGF optimally increased the cell number by 1.8-fold at 1.0 ng/ml for a 4-day incubation period under subconfluent culture. In contrast, under confluent culture, 10 ng/ml HB-EGF optimally increased the DNA synthesis <em>2</em>.1-fold. To examine the production of HB-EGF by human <em>keratinocytes</em>, the analysis of human <em>keratinocyte</em>-conditioned medium was undertaken by a combination of heparin affinity column chromatography, EGF receptor-stimulating assay, immunoblotting, and neutralization. Heparin column chromatography fractionated three activities, peaks 1, <em>2</em>, and 3, which contained immunoreactive 30- and <em>2</em>7-, 19-, and 14.5-kDa bands, respectively. The anti-HB-EGF-blocking antibody neutralized the activities of peaks <em>2</em> and 3 by 38 and <em>2</em><em>2</em>%, respectively, but did not neutralize the activity of peak 1 at all. The antibody reduced the cell <em>growth</em> by 37% for a 4-day incubation period. Northern blot analysis detected a <em>2</em>.5-kilobase transcript of HB-EGF. The addition of 1 ng/ml HB-EGF optimally increased the levels of HB-EGF mRNA 5.4-fold at 1 h and TGF-alpha mRNA 3.1-fold at 3 h. Interestingly, the addition of TGF-alpha at 1 ng/ml to <em>keratinocyte</em> cultures enhanced the level of HB-EGF mRNA 10.<em>2</em>-fold at 6 h. 1 ng/ml EGF also increased HB-EGF mRNA levels 10.9-fold at 1 h. These results suggest that HB-EGF is an autocrine <em>growth</em> <em>factor</em> for human <em>keratinocytes</em>, and HB-EGF and TGF-alpha act not only by an autoinductive mechanism but also by mutual amplification.

Calcium functions as a trigger for the switch between epithelial cell <em>growth</em> and differentiation. We report here that the calcium/calmodulin-dependent phosphatase calcineurin is involved in this process. Treatment of primary mouse <em>keratinocytes</em> with cyclosporin A, an inhibitor of calcineurin activity, suppresses the expression of terminal differentiation markers and of p<em>2</em>1(WAF1/Cip1) and p<em>2</em>7(KIP1), two cyclin-dependent kinase inhibitors that are usually induced with differentiation. In parallel with down-modulation of the endogenous genes, suppression of calcineurin function blocks induction of the promoters for the p<em>2</em>1(WAF1/Cip1) and loricrin differentiation marker genes, whereas activity of these promoters is enhanced by calcineurin overexpression. The calcineurin- responsive region of the p<em>2</em>1 promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA box, and calcineurin induces activity of the p<em>2</em>1 promoter through Sp1/Sp3-dependent transcription. We find that the endogenous NFAT-1 and -<em>2</em> transcription <em>factors</em>, major downstream targets of calcineurin, associate with Sp1 in <em>keratinocytes</em> in a calcineurin-dependent manner, and calcineurin up-regulates Sp1/Sp3-dependent transcription and p<em>2</em>1 promoter activity in synergism with NFAT1/<em>2</em>. Thus, our study reveals an important role for calcineurin in control of <em>keratinocyte</em> differentiation and p<em>2</em>1 expression, and points to a so-far-unsuspected interconnection among this phosphatase, NFATs, and Sp1/Sp3-dependent transcription.

IL-17C is a functionally distinct member of the IL-17 family that binds IL-17 receptor E/A to promote innate defense in epithelial cells and regulate Th17 cell differentiation. We demonstrate that IL-17C (not IL-17A) is the most abundant IL-17 isoform in lesional psoriasis skin (1058 versus 8 pg/ml; p < 0.006) and localizes to <em>keratinocytes</em> (KCs), endothelial cells (ECs), and leukocytes. ECs stimulated with IL-17C produce increased TNF-α and KCs stimulated with IL-17C/TNF-α produce similar inflammatory gene response patterns as those elicited by IL-17A/TNF-α, including increases in IL-17C, TNF-α, IL-8, IL-1α/β, IL-1F5, IL-1F9, IL-6, IL-19, CCL<em>2</em>0, S100A7/A8/A9, DEFB4, lipocalin <em>2</em>, and peptidase inhibitor 3 (p < 0.05), indicating a positive proinflammatory feedback loop between the epidermis and ECs. Psoriasis patients treated with etanercept rapidly decrease cutaneous IL-17C levels, suggesting IL-17C/TNF-α-mediated inflammatory signaling is critical for psoriasis pathogenesis. Mice genetically engineered to overexpress IL-17C in KCs develop well-demarcated areas of erythematous, flakey involved skin adjacent to areas of normal-appearing uninvolved skin despite increased IL-17C expression in both areas (p < 0.05). Uninvolved skin displays increased angiogenesis and elevated S100A8/A9 expression (p < 0.05) but no epidermal hyperplasia, whereas involved skin exhibits robust epidermal hyperplasia, increased angiogenesis and leukocyte infiltration, and upregulated TNF-α, IL-1α/β, IL-17A/F, IL-<em>2</em>3p19, vascular endothelial <em>growth</em> <em>factor</em>, IL-6, and CCL<em>2</em>0 (p < 0.05), suggesting that IL-17C, when coupled with other proinflammatory signals, initiates the development of psoriasiform dermatitis. This skin phenotype was significantly improved following 8 wk of TNF-α inhibition. These findings identify a role for IL-17C in skin inflammation and suggest a pathogenic function for the elevated IL-17C observed in lesional psoriasis skin.

The stromal microenvironment plays a crucial role in tumor development and progression. One of the most potent activators of stromal cells is the platelet-derived <em>growth</em> <em>factor</em> (PDGF). To investigate the role of PDGF in epithelial tumor development we stably transfected immortal nontumorigenic human <em>keratinocytes</em> with the PDGF-B cDNA. Transfected HaCaT cells overexpressed PDGF-B but remained negative for the PDGF receptors alpha and beta (mRNA). Thus, they did not exhibit autocrine <em>growth</em> stimulation in vitro, but proliferation of cocultured fibroblasts was enhanced and this effect was inhibited by a neutralizing antibody to PDGF-BB. After subcutaneous injection into nude mice the transfected cells maintained high PDGF expression and formed progressively enlarging, rapidly proliferating cysts, classified as benign tumors. During early tumor development (up to <em>2</em> months), PDGF-B transfectants induced marked mesenchymal cell proliferation and angiogenesis, yet this effect vanished at later stages (<em>2</em>-6 months) concomitantly with increased epithelial cell proliferation and enhanced tumor <em>growth</em>. These results demonstrate that an activated stromal environment can promote tumorigenic conversion of nontumorigenic <em>keratinocytes</em> by inducing sustained epithelial hyperproliferation. This effect is apparently caused by a dual action of PDGF-BB: (i) PDGF-BB can promote tumor <em>growth</em> by inducing angiogenesis and stroma formation, and (ii) PDGF-activated stromal cells maintain elevated <em>keratinocyte</em> proliferation via a paracrine mechanism. Thus, PDGF, a major <em>factor</em> activated in wound healing, may play an important role as an endogenous promoter in epithelial tumor formation.

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 1<em>2</em>, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L<em>2</em> (cathepsin V), uroplakin Ib, and Ca(<em>2</em>+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, <em>growth</em> <em>factors</em> and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main <em>factors</em> being epidermal <em>growth</em> <em>factor</em> (EGF), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), hepatocyte <em>growth</em> <em>factor</em> (HGF), transforming <em>growth</em> <em>factor</em>-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.

The Jun proteins Jun, JunB and JunD are core members of activator protein-1 (AP-1), a dimeric transcription <em>factor</em> complex consisting of homo- and heterodimers of the Jun, Fos, activating transcription <em>factor</em> (ATF) and musculoaponeurotic fibrosarcoma (Maf) families. <em>Growth</em> <em>factors</em>, hormones and a variety of environmental stresses activate mitogen activated protein kinase (MAPK) cascades that enhance Jun/AP-1 activity, e.g. through phosphorylation thereby regulating cell proliferation, differentiation, transformation and/or apoptosis. Embryonic lethality of various AP-1 knock-outs, e.g. for Jun, JunB, Fra-1 and Fra-<em>2</em> largely prevented functional studies in vivo. Therefore, conditional knock-out strategies, in particular for the epidermis, have become an important model to study the regulation and function of AP-1 subunits in physiological and pathological processes in vivo. Jun is regarded as a positive regulator of <em>keratinocyte</em> proliferation/differentiation during development and in skin cancer through its direct transcriptional effect on epidermal <em>growth</em> <em>factor</em> receptor (EGFR) expression. In contrast, JunB can antagonize proliferation of <em>keratinocytes</em> and hematopoietic stem cells. Furthermore, it has been demonstrated in patient's samples and an inducible mouse model that down-regulation of JunB/AP-1 in <em>keratinocytes</em> is one initiating event in the aetiology of psoriasis which is characterized by increased cell proliferation and deregulated cytokine expression.