Vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A, a key regulator of cutaneous blood vessel formation, appears to have an additional role during wound healing, enhancing re-epithelialization. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that initiate in wounds and are characterized by extensive blood vessel formation, epidermal hyperplasia and rete ridge formation. The vascular changes beneath the lesion are largely due to viral-expressed VEGF-E. This study investigated using mouse skin models whether VEGF-E can induce epidermal changes such as that seen in the viral lesion. Injection of VEGF-E into normal skin increased the number of endothelial cells and blood vessels within the dermis and increased epidermal thickening and <em>keratinocyte</em> number. Injection of VEGF-E into wounded skin, which more closely mimics orf virus lesions, increased neo-epidermal thickness and area, promoted rete ridge formation, and enhanced wound re-epithelialization. Quantitative RT-PCR analysis showed that VEGF-E did not induce expression of epidermal-specific <em>growth</em> <em>factors</em> within the wound, but did increase matrix metalloproteinase (MMP)-<em>2</em> and MMP-9 expression. In cell-based assays, VEGF-E induced <em>keratinocyte</em> migration and proliferation, responses that were inhibited by a neutralizing antibody against VEGF receptor (VEGFR)-<em>2</em>. These findings demonstrate that VEGF-E, both directly and indirectly, regulates <em>keratinocyte</em> function, thereby promoting epidermal regeneration.

Reprogramming differentiated cells toward stem cells may have long-term applications in stem-cell research and regenerative medicine. Here we report on the dedifferentiation of human epidermal <em>keratinocytes</em> into their precursor cells in vitro with basic fibroblast <em>growth</em> <em>factor</em> (bFGF) but not external gene intervention. After incubation of human terminally differentiating <em>keratinocytes</em>, some of the surviving <em>keratinocytes</em> reverted from a differentiated to a dedifferentiated state, as evidenced by re-expression of biological markers of native <em>keratinocyte</em> stem cells (nKSCs), including β(1)-integrin, CK19 and CK14. Moreover, these dedifferentiation-derived KSCs (dKSCs) showed an ability for high colony formation correlated with cell cycle analysis showing a marked accumulation in S phases, acquired a similar regional distribution of both α(6)-integrin and CD71 expression at the ultrastructural level, and had a increased proliferative capacity by releasing telomerase from nucleolar sites to nucleoplasmic distribution. However, on comparing dKSCs with nKSCs, <em>2</em> points seem noteworthy: (1) the proportion of transit amplifying cells in dKSCs treated with bFGF is much higher than that in nKSCs and (<em>2</em>) regional differences exist in the subcellular localization of telomerase in nKSCs and dKSCs. Most nKSCs showed a prominent nucleolar concentration of human telomerase reverse transcriptase expression, whereas most dKSCs showed a more diffuse intranuclear distribution of telomerase or even signal depletion at nucleoli relative to the general nucleoplasm. These results indicate that bFGF could induce the terminally differentiating epidermal <em>keratinocytes</em> to convert into their precursor cells, which offers a new approach for generating residual healthy stem cells for wound repair and regeneration.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, <em>2</em>, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-<em>2</em>, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-<em>2</em>, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days <em>2</em> (<em>2</em>0-40%) and 3 (0-10%), and reappeared at day 7 (<em>2</em>5-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-<em>2</em>. High Bcl-<em>2</em>:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-<em>2</em>:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.

The structural gene for human <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), a member of the fibroblast <em>growth</em> <em>factor</em> family, consists of three coding exons and two introns typical of other fibroblast <em>growth</em> <em>factor</em> loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon <em>2</em>, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.

<em>Growth</em> <em>factors</em> play a key role in cellular communication, a necessary step for the development of pluricellular organisms. The fibroblast <em>growth</em> <em>factors</em> (FGF) are among these polypeptides and have seven known members: FGF 1 to FGF 7 which are also known as acidic FGF, basic FGF, translation products of oncogenes hst, int <em>2</em>, FGF 5, FGF 6 and FGF 7 or <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) respectively [1]. The best known and the most abundant in normal adult tissues are acidic and basic FGFs, or FGF 1 and <em>2</em> respectively, which have been subjected to extensive studies both in vitro and in vivo. These two <em>factors</em> have almost ubiquitous distribution and a wide spectrum of biological activity including action on cellular proliferation and differentiation, as well as neurotrophic and angiogenic properties [1]. These different activities are induced by triggering specific receptors present at the surface of the target cell. Following this interaction, the FGF-receptor complexes are internalized and activate intracellular pathways. An important effort of investigations has been produced to characterize these receptors and intracellular pathways. It is the purpose of this review to present this work which will focus on FGFs 1 and <em>2</em>. The existence of two classes of interactions has been reported as early as 1987 [5<em>2</em>, 53, 54] suggesting the presence of high and low affinity receptors for FGFs.

Cell-cell communication is necessary for the crosstalk between cells that constitute multicellular organisms and is essential for cells to coordinate their physiological behavior to create cohesive tissues. Cellular crosstalk is not only controlled by molecules, like <em>growth</em> <em>factors</em>, hormones, ions and G-proteins, etc. but also by cell-cell contacts. These contacts are essential for intercellular communication and are involved in survival, apoptosis, proliferation, differentiation and homeostasis of entire tissues. In polarized epithelia of vertebrates, the adherent junction is part of the tripartite junctional complex that is localized at the juxtaluminal region, which includes tight junctions (including claudins, occludins, and zonula occludens proteins), desmosomal junctions (including desmogleins), and adherent junctions. In focus of the manuscript are adherent molecules of the cadherin superfamily of the skin. In the normal epidermis, melanocytes and <em>keratinocytes</em> are mostly connected via E-cadherin, P-cadherin and H-cadherin [1-3]. Melanocytes that reside in the basal layer of the epidermis predominantly contain E-cadherin and H-cadherin, whereas those that reside in the hair follicles are rich in P-cadherin [<em>2</em>]. The regulation and role of E-cadherin during melanoma development will be the focus of this review.

Reactive oxygen species (ROS) increase permeability in the airway epithelium. Extended periods of oxidant exposure may be experienced by those suffering from chronic inflammation of the lungs, receiving supplemental oxygen, or living in areas with high levels of air pollution. We studied the effects of long-term, continuous exposure to hydrogen peroxide (H(<em>2</em>)O(<em>2</em>)) on the trans-epithelial electrical resistance (TER) across cultured monolayers of a transformed cell line of human bronchial epithelial cells, 16HBE14o- (16HBE). A TER perfusion system was employed to continuously monitor the TER without disturbing the tissue model. The TER decreased in a dose-dependent manner with increasing concentrations of H(<em>2</em>)O(<em>2</em>) (0.1, 0.5, and 1.0 mM), regardless of pre-incubation conditions. Cell cultures pre-treated with 50 ng/ml <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) showed a significant delay in oxidant-induced TER decreases caused by 0.1 mM H(<em>2</em>)O(<em>2</em>). Exposure to 0.1 mM H(<em>2</em>)O(<em>2</em>) for 350 min led to disruption of tight junction proteins, ZO-1 and occludin, but KGF treatment prevented this damage. The recovery of epithelial barrier function after exposure to oxidants was also studied. Tissue models exposed to 0.5 mM H(<em>2</em>)O(<em>2</em>) for <em>2</em>5 min showed complete recovery of TER after <em>2</em>0 h, independent of culture pre-treatment. In contrast, KGF pre-incubation enhanced the recovery of 16HBE cultures exposed for 50 min to 0.5 mM H(<em>2</em>)O(<em>2</em>).

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4(+)Foxp3(+) Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4(+)Foxp3(+) Treg. Blood Treg numbers remained elevated for>><em>2</em> mo, but the frequency normalized after <em>2</em> wk due to a concomitant increase in CD4(+)Foxp3(-) T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4(+) T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4(+)Foxp3(+) Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.

The hepatocyte <em>growth</em> <em>factor</em> (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer <em>growth</em>. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, "v5") of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic <em>keratinocyte</em> (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/<em>2</em> MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-<em>2</em>31 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-<em>2</em>31 cell migration occurred independently of ERK1/<em>2</em> and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.

Epidermal <em>keratinocyte</em> cultures were established from newborn mice expressing a null mutation in the p53 gene to explore the contribution of p53 to epidermal <em>growth</em> regulation and neoplasia. <em>Keratinocytes</em> were initiated by transduction with a replication-defective retrovirus encoding the v-rasHa oncogene and grafted onto nude mouse hosts. Tumors arising from <em>keratinocytes</em> heterozygous or null for functional p53 in the presence of v-rasHa have <em>growth</em> rates approximately 5-fold higher than those derived from p53(+/+) controls and rapidly form carcinomas, in contrast to the benign phenotype observed in p53(+/+)/v-rasHa grafts. In vitro, p53-deficient <em>keratinocytes</em> with and without v-rasHa expression display decreased responsiveness to the negative <em>growth</em> regulators transforming <em>growth</em> <em>factors</em> beta 1 and beta <em>2</em>. In combination with v-rasHa, p53-deficient <em>keratinocytes</em> also exhibit decreased responsiveness to elevated Ca<em>2</em>+. These differences between genotypes cannot be attributed to changes in transforming <em>growth</em> <em>factor</em> beta receptor types present or altered levels of epidermal <em>growth</em> <em>factor</em> receptor and are independent of c-myc transcript levels. mRNA expression for the p-53 inducible protein WAF1 correlates with p53 gene dosage, but low levels are still detectable in p53(-/-) <em>keratinocytes</em>. The altered responsiveness of p53 deficient <em>keratinocytes</em> to negative <em>growth</em> regulators may provide a <em>growth</em> advantage to such cells in vivo and render them more susceptible to genetic alterations and malignant conversion.

BACKGROUND

Inadequate healing and consequent leakage from bowel anastomoses are a significant cause of postoperative morbidity and mortality. Systemic application of keratinocyte growth factor (KGF) has been shown to promote mucosal healing in models of colitis in rats and mice. The aim of the present study was to evaluate the effect of systemic KGF administration on healing of colonic anastomoses in rats.

METHODS

Rats underwent laparotomy, division of the left colon, and sigmoido-sigmoidostomy. KGF (5 mg/kg) or vehicle were administered intraperitoneally in two groups (n = 30 per group) 12 hours prior to surgery, and then once daily until sacrifice (6 animals per group; 2, 4, 7, 12, and 21 days after surgery). Bursting pressure measurements, histologic evaluation, morphometric analysis, mucin and collagen staining, and hydroxyproline measurements of the anastomotic site were performed.

RESULTS

Administration of KGF significantly increased anastomotic bursting pressure on postoperative days 2, 4, and 7 by 34%, 49%, and 19%, respectively. Histology, mucin staining, and measurements of the colonic crypt depth showed markedly less extended inflammation with an increased acidic mucin content and a significantly thickened mucosal layer in the KGF treated group when compared with vehicle-treated animals.

CONCLUSIONS

KGF promotes healing of colonic anastomoses in rats during a 1-week postoperative period following large bowel surgery. KGF may be acting to accelerate host reparative processes as well as to enhance protection of the anastomotic wound bed by increased colonic epithelium proliferation, increased mucus production, and reduction of the inflammatory activity at the anastomotic site.

Transforming <em>growth</em> <em>factor</em> beta (TGF-beta) inhibits proliferation of normal <em>keratinocytes</em>, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta <em>2</em>, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 1<em>2</em>-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal <em>keratinocytes</em>, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 1<em>2</em>-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal <em>keratinocyte</em> compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative <em>growth</em> regulation by altered translational and/or posttranslational processing mechanisms of this <em>growth</em> <em>factor</em>. Levels of TGF-beta <em>2</em> and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.

Mesothelial cells proliferate soon after asbestos deposition in the lung. The present study investigates whether the known mesothelial cell mitogens <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) are present in the lung and specifically in the pleural cavity during the phase of mesothelial cell <em>growth</em>. Rats received 1 mg crocidolite asbestos in 0.5 mL water by intratracheal instillation and were killed up to <em>2</em> weeks later; tritiated thymidine was injected 1 hour before death. Bronchoalveolar lavage (BAL) and pleural lavage (PLL) were performed. Increased inflammatory cell numbers and protein levels were found in BAL but also in PLL at 1 day after asbestos deposition. In lung sections, labeling of mesothelial cells increased>> 10-fold at day 1 and stayed above normal for 1 week. During this period, the levels of HGF and KGF were significantly raised in both BAL and PLL fluids. The PLL fluid had mitogenic activity for mesothelial cells in culture and this effect was significantly reduced by antibodies to HGF and KGF. The results indicate that fiber deposition in the airspaces rapidly induces lung injury and inflammation, in which <em>growth</em> <em>factors</em> for mesothelial cells KGF and HGF are secreted. These <em>factors</em> reach the pleural cavity at the time when mesothelial cell proliferation occurs. It is possible that the activated, dividing mesothelial population may then be more susceptible to DNA damage by any translocated fibers.

BACKGROUND

l-Ascorbic acid <em>2</em>-phosphate (Asc <em>2</em>-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair <em>growth</em> in mice.

OBJECTIVE

To investigate whether the promotion of hair <em>growth</em> by Asc <em>2</em>-P is mediated by insulin-like <em>growth</em> factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc <em>2</em>-P-induced IGF-1 expression.

METHODS

Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc <em>2</em>-P treatment in the absence or presence of LY<em>2</em>9400<em>2</em>, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc <em>2</em>-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc <em>2</em>-P treatment in the absence or presence of LY<em>2</em>9400<em>2</em> was examined by Ki-67 immunostaining.

RESULTS

IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc <em>2</em>-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc <em>2</em>-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc <em>2</em>-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc <em>2</em>-P-induced <em>growth</em> of cocultured keratinocytes. LY<em>2</em>9400<em>2</em> significantly attenuated Asc <em>2</em>-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles.

CONCLUSIONS

These data show that Asc <em>2</em>-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle <em>growth</em> in vitro via PI3K.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) is one of the most abundant <em>growth</em> <em>factors</em> secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and <em>growth</em>. Previous studies from our laboratory demonstrated that 1 alpha, <em>2</em>5-dihydroxyvitamin D3 (1 alpha,<em>2</em>5(OH)<em>2</em>D3) inhibits human osteoblast and <em>keratinocyte</em> <em>growth</em> by increasing TGF-beta<em>2</em> secretion and synthesis. To examine the mechanism by which 1 alpha,<em>2</em>5(OH)<em>2</em>D3 regulates TGF-beta <em>2</em> transcription in osteoblasts, we ligated segments of the human TGF-beta <em>2</em> promoter 5' of a <em>growth</em> hormone reporter gene in a <em>growth</em> hormone reporter plasmid and examined the effects of 1 alpha,<em>2</em>5(OH)<em>2</em>D3 administration on the expression of <em>growth</em> hormone in cells transfected with such chimeric promoter-reporter plasmids. The promoter region extending from -973 to +68 bp of the transcription start site elicited a 7-fold increase in reporter gene activity in transiently transfected osteoblasts after 1 alpha,<em>2</em>5(OH)<em>2</em>D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,<em>2</em>5(OH)<em>2</em>D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta <em>2</em> promoter DNA from -869 to -658 bp revealed a 3.8-fold increase in reporter gene activity. DNA fragments from this region (-743 to -676 bp and -786 to -7<em>2</em>8 bp) ligated into reporter plasmids also showed increases in reporter activity. Gel retardation assay experiments showed that DNA fragments from -774 to -735 bp and -714 to -675 bp both formed a DNA-protein complex with bacterially expressed human retinoic acid X receptor alpha (RXR alpha) and vitamin D receptor (VDR) and with nuclear extracts from human bone cells. Addition of a vitamin D receptor antibody to reactions containing the aforesaid DNA fragments and bone cell nuclear extract resulted in further retardation of the mobility of the DNA-protein complex (super-shifting). Removal of two putative direct repeat DNA fragments in this region abolished VDR-RXR alpha-vitamin D response element complex formation. The TGF-beta <em>2</em> promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,<em>2</em>5(OH)<em>2</em>D3.

Bone morphogenetic proteins (BMPs), members of the transforming <em>growth</em> <em>factor</em>-beta family, signal in many cells including neural precursors. Two receptors, types 1 and <em>2</em>, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -<em>2</em>. Furthermore, melanocytes and the surrounding <em>keratinocytes</em> express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a member of the fibroblast <em>growth</em> <em>factor</em> family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15-<em>2</em>1.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons <em>2</em> and 3 were localized to dispersed sites on chromosome <em>2</em>q<em>2</em>1, 9p11, 9q1<em>2</em>-13, 18p11, 18q11, <em>2</em>1q11, and <em>2</em>1q<em>2</em>1.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and <em>2</em>1. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (<em>2</em>.4 U/ml) and in deoxyribonuclease (0.0<em>2</em>%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbecco's modified Eagle's medium and Ham's F 1<em>2</em> medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal <em>growth</em> <em>factor</em> (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO<em>2</em>-incubator at 37 degrees C. After <em>2</em>-3 wk cell out<em>growth</em>s resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of <em>keratinocytes</em> in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.

Skin <em>keratinocytes</em> express tissue <em>factor</em> (TF) and are highly associated with skin wound healing. Although it has been demonstrated that perivascular TF expression in granulation tissue formed after dermal injury is downregulated during healing, studies of the mechanism of <em>factor</em> (F) VII, a TF ligand, in skin wound healing are lacking. We reported the use of a dermal punch model to demonstrate that low-expressing FVII mice (approximately 1% of wild type [WT]) exhibited impaired skin wound healing compared with WT controls. These low-FVII mice showed defective reepithelialization and reduced inflammatory cell infiltration at wound sites. This attenuated reepithelialization was associated with diminished expression of the transcription <em>factor</em> early <em>growth</em> response 1 (Egr-1). In vitro, Egr-1 was shown to be essential for the FVIIa-induced regulation of <em>keratinocyte</em> migration and inflammation. Both Egr-1 upregulation and downstream inflammatory cytokine appearance in <em>keratinocytes</em> depended on FVIIa/TF/protease-activated receptor <em>2</em> (PAR-<em>2</em>)-induced signaling and did not require subsequent generation of FXa and thrombin. The participation of Egr-1 in FVIIa-mediated regulation of <em>keratinocyte</em> function was confirmed by use of Egr-1-deficient mice, wherein a significant delay in skin wound healing after injury was observed, relative to WT mice. The results from these studies demonstrate an in vivo mechanistic relationship between FVIIa, Egr-1 and the inflammatory response in <em>keratinocyte</em> function during the wound healing process.

BACKGROUND

Inorganic arsenic is an environmental contaminant and is associated with the increased risk of human skin cancer. Arsenic has been reported to activate or inhibit a variety of cellular signalling pathways which has effects on cell growth, differentiation and apoptosis. However, the molecular mechanisms of these arsenic-induced biological effects are not completely understood.

OBJECTIVE

To understand the molecular basis for the mode of action of arsenicals, we examined the effect of arsenite and arsenate on the activation of mitogen-activated protein kinases (MAPK) and the upstream signalling cascade in normal human epidermal keratinocytes (NHEK).

METHODS

NHEK were exposed to arsenite or arsenate. Western blot analysis was performed to determine the activation of extracellular signal-regulated kinases (ERK) 1/2, c-jun N-terminal kinases (JNK), p38, and MAPK or ERK kinases (MEK) 1/2. Epidermal growth factor receptor (EGFR) tyrosine phosphorylation and recruitment of its adaptor proteins, Shc and Grb2, to EGFR were detected by immunoprecipitation and Western blot analysis.

RESULTS

Both arsenicals activated ERK1/2, which are most highly activated in response to mitogenic stimulation, in addition to JNK and p38, which show greater activation in response to cellular stresses. The kinetics of ERK1/2 activation differed from those of JNK and p38 activation. Both arsenicals transiently activated ERK1/2 prior to JNK and p38 activation. MEK1/2, upstream kinases of ERK1/2, were also activated by arsenicals with similar time kinetics to that of ERK1/2 activation. To investigate a signalling pathway leading to activation of MEK1/2-ERK1/2, we examined the tyrosine phosphorylation of EGFR and Shc adapter protein. Both arsenicals stimulated tyrosine phosphorylation of EGFR and Shc. After arsenical treatment, Shc immunoprecipitates contained coprecipitated EGFR and Grb2, suggesting that both arsenicals induce the assembly of EGFR-Shc-Grb2 complexes. Both the EGFR inhibitor tyrphostin AG1478 and anti-EGFR blocking antibody markedly attenuated ERK1/2 activation induced by arsenicals, but did not affect JNK and p38 activation.

CONCLUSIONS

Our data indicate that both arsenite and arsenate activate the EGFR-Shc-Grb2-MEK1/2-ERK1/2 signalling cascade in NHEK.

Interleukin-15 (IL-15) is a recently discovered cytokine produced by a wide range of different cell types including fibroblasts, <em>keratinocytes</em>, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-15 may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-15, evaluated as enhancement of PMN anti-Candida activity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-15 depends on binding to the IL-15 receptor. Our experiments show that binding of a biotinylated human IL-15-immunoglobulin G<em>2</em>b IgG<em>2</em>b fusion protein was competed by the addition of human recombinant IL-15 (rIL-15) or of human rIL-<em>2</em>, suggesting that IL-15 binding to PMN might involve the IL-<em>2</em>Rbeta and IL-<em>2</em>Rgamma chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-15Ralpha chain monoclonal antibody that PMN express the IL-15Ralpha chain at the mRNA and protein levels. Incubation with IL-15 activated PMN to secrete the chemotactic <em>factor</em> IL-8, and the amount secreted was increased by costimulation with heat-inactivated Candida albicans. In addition, IL-15 primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed to C. albicans. IL-15 also increased the ability of PMN to phagocytose heat-killed C. albicans organisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-15 was as effective as gamma interferon (IFN-gamma) and IL-<em>2</em> in increasing the C. albicans <em>growth</em>-inhibitory activity of PMN. Taken together, these results suggest that IL-15 is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response against C. albicans.

AW464 (NSC 706704) is a novel benzothiazole substituted quinol compound active against colon, renal and certain breast cancer cell lines. NCI COMPARE analysis indicates possible interaction with thioredoxin/thioredoxin reductase, which is upregulated under hypoxia. Through activity on HIF1alpha, VEGF levels are regulated and angiogenesis controlled. A thioredoxin inhibitor could therefore exhibit enhanced hypoxic toxicity and indirect antiangiogenic effects. In vitro experiments were performed on colorectal and breast cancer cell lines under both normoxic and hypoxic conditions and results compared against those obtained with normal cell lines, fibroblasts and <em>keratinocytes</em>. Antiangiogenic effects were studied using both large and microvessel cells. Indirect antiangiogenic effects (production of angiogenic <em>growth</em> <em>factors</em>) were studied via ELISA. We show that AW464 exerts antiproliferative effects on tumour cell lines as well as endothelial cells with an IC(50) of approximately 0.5 microM. Fibroblasts are however resistant. Proliferating, rather than quiescent, endothelial cells are sensitive to the drug indicating potential antiangiogenic rather than antivascular action. Endothelial differentiation is also inhibited in vitro. Hypoxia (1% O(<em>2</em>) for 48 h) sensitises colorectal cells to lower drug concentrations, and in HT<em>2</em>9s greater inhibition of VEGF is observed under such conditions. In contrast, bFGF levels are unaffected, suggesting possible involvement of HIF1alpha. Thus, AW464 is a promising chemotherapeutic drug that may have enhanced potency under hypoxic conditions and also additional antiangiogenic activity.

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human <em>keratinocyte</em> stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the <em>keratinocyte</em> progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast <em>growth</em> <em>factor</em> type <em>2</em> (FGF<em>2</em>) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF<em>2</em> protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF<em>2</em> in DNA repair, stem cells were exposed to FGF<em>2</em> pathway inhibitors. Blocking the FGF<em>2</em> receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the <em>keratinocyte</em> stem cells. Moreover, supplementing the progenitor cells with exogenous FGF<em>2</em> activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival <em>factor</em>, FGF<em>2</em> helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.

In addition to its known effects on <em>keratinocyte</em> proliferation and differentiation, the hormonal form of vitamin D, 1,<em>2</em>5-dihydroxyvitamin D(3) (1,<em>2</em>5(OH)(<em>2</em>)D(3)), has been shown to protect <em>keratinocytes</em> from UV- and chemotherapy-induced damage. Epidermal <em>keratinocytes</em> contain both the machinery needed to produce 1,<em>2</em>5(OH)(<em>2</em>)D(3) and vitamin D receptors. The activation of the stress-activated protein kinases (SAPKs), such as c-Jun N-terminal kinase (JNK) and p38, is an early cellular response to stress signals and an important determinant of cell fate. This study examines whether modulation of these SAPKs is associated with the effects of 1,<em>2</em>5(OH)(<em>2</em>)D(3) on <em>keratinocytes</em> under stress. HaCaT <em>keratinocytes</em> were exposed to heat shock, hyperosmotic concentrations of sorbitol, the epidermal <em>growth</em> <em>factor</em> receptor tyrosine kinase inhibitor AG1487, the pro-inflammatory cytokine tumor necrosis <em>factor</em> alpha, and H(<em>2</em>)O(<em>2</em>). These stresses activated both SAPKs. Pretreatment with 1,<em>2</em>5(OH)(<em>2</em>)D(3) inhibited the activation of JNK by all stresses and the activation of p38 by heat shock, AG1478 and tumor necrosis <em>factor</em> alpha. Under the same conditions, treatment with 1,<em>2</em>5(OH)(<em>2</em>)D(3) protected HaCaT <em>keratinocytes</em> from cytotoxicity induced by exposure to H(<em>2</em>)O(<em>2</em>) and hyperosmotic shock. The effect of 1,<em>2</em>5(OH)(<em>2</em>)D(3) was dose-dependent, already apparent at nanomolar concentrations, and time-dependent, maximal after a <em>2</em>4-h pre-incubation. We suggest that inhibition of SAPK activation may account for some of the well-documented protective effects of 1,<em>2</em>5(OH)(<em>2</em>)D(3) on epidermal cells during exposure to UV or chemotherapy and may also be related to the anti-inflammatory actions of the hormone in skin.