BACKGROUND

Although some patients with psoriasis vulgaris also complain of severe pruritus, the data available regarding pruritus in psoriasis are sparse.

OBJECTIVE

To clarify the mechanism and mediators involved in the pruritus of psoriasis vulgaris, we compared itch-associated factors in lesional skin from psoriatic patients vs. skin without pruritus quantitatively using a panel of histological and immunohistological parameters.

METHODS

Biopsied specimens were obtained from 38 patients with psoriasis vulgaris who were divided into two groups according to the presence or absence of pruritus.

RESULTS

When compared with psoriatic patients devoid of pruritus, lesional skin from patients with pruritus showed the following characteristic features: (i) a rich innervation both in the epidermis and in the papillary dermis; (ii) an increase in neuropeptide substance P-containing nerve fibres in perivascular areas; (iii) decreased expression of neutral endopeptidase in the epidermal basal layer as well as in the endothelia of blood vessels; (iv) many mast cells showing degranulating processes in the papillary dermis; (v) a strong immunoreactivity for nerve growth factor (NGF) throughout the entire epidermis and an increased NGF content in lesional skin homogenates; (vi) an increase in the expression of high-affinity receptors for NGF (Trk A) in basal keratinocytes and in dermal nerves; (vii) an increased population of interleukin-2-immunoreactive lymphocytes; and (viii) a strong expression of E-selectin on vascular endothelial cells. A significant correlation was observed between the severity of pruritus and protein gene product 9.5-immunoreactive intraepidermal nerve fibres, NGF-immunoreactive keratinocytes, expression of Trk A in the epidermis and the density of immunoreactive vessels for E-selectin. These findings indicate that possible pruritogenic mediators in psoriatic lesional skin are neurogenic factors including innervation, neuropeptide substance P, neuropeptide-degrading enzymes and NGF, activated mast cells, one or more cytokines and endothelial-leucocyte adhesion molecules.

CONCLUSIONS

These data document for the first time itch-related local markers in psoriasis, and suggest complex and multifactorial mechanisms of pruritus in the disease. These results provide the groundwork for further studies to evaluate the efficacy of antipruritic treatment for psoriatic patients.

Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived <em>growth</em> <em>factor</em>-BB and transforming <em>growth</em> <em>factor</em>-β1, in the presence of fibroblast <em>growth</em> <em>factor</em> <em>2</em>, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic <em>keratinocytes</em>, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-1<em>2</em>6, miR-130a, miR-13<em>2</em>) and anti-inflammatory (miR1<em>2</em>4a, miR-1<em>2</em>5b) microRNAs, and a microRNA regulating collagen deposition (miR-<em>2</em>1). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers.

The fibroblast <em>growth</em> <em>factor</em>-binding protein (FGF-BP) binds and activates FGF-1 and FGF-<em>2</em>, thereby contributing to tumor angiogenesis. In this study, we identified novel binding partners of FGF-BP, and we provide evidence for a role of this protein in epithelial repair processes. We show that expression of FGF-BP increases after injury to murine and human skin, in particular in <em>keratinocytes</em>. This upregulation is most likely achieved by major <em>keratinocyte</em> mitogens present at the wound site. Most importantly, we demonstrate that FGF-BP interacts with FGF-7, FGF-10, and with the recently identified FGF-<em>2</em><em>2</em>, and enhances the activity of low concentrations of ligand. Due to the important functions of FGF-7 and FGF-10 for repair of injured epithelia, our findings suggest that upregulation of FGF-BP expression after injury stimulates FGF activity at the wound site, thus enhancing the process of epithelial repair.

OBJECTIVE

Glucagon-like peptide-<em>2</em> (GLP-<em>2</em>) is a 33-amino acid peptide hormone secreted by enteroendocrine cells in response to nutrient ingestion. GLP-<em>2</em> stimulates crypt cell proliferation leading to expansion of the mucosal epithelium; however, the mechanisms transducing the trophic effects of GLP-<em>2</em> are incompletely understood.

METHODS

We examined the gene expression profiles and growth-promoting actions of GLP-<em>2</em> in normal mice in the presence or absence of an inhibitor of ErbB receptor signaling, in Glp<em>2</em>r(-/-) mice and in Egfr(wa<em>2</em>) mice harboring a hypomorphic point mutation in the epidermal growth factor receptor.

RESULTS

Exogenous GLP-<em>2</em> administration rapidly induced the expression of a subset of ErbB ligands including amphiregulin, epiregulin, and heparin binding (HB)-epidermal growth factor, in association with induction of immediate early gene expression in the small and large bowel. These actions of GLP-<em>2</em> required a functional GLP-<em>2</em> receptor because they were eliminated in Glp<em>2</em>r(-/-) mice. In contrast, insulin-like growth factor-I and keratinocyte growth factor, previously identified mediators of GLP-<em>2</em> action, had no effect on the expression of these ErbB ligands. The GLP-<em>2</em>-mediated induction of ErbB ligand expression was not metalloproteinase inhibitor sensitive but was significantly diminished in Egfr(wa<em>2</em>) mice and completed abrogated in wild-type mice treated with the pan-ErbB inhibitor CI-1033. Furthermore, the stimulatory actions of GLP-<em>2</em> on crypt cell proliferation and bowel growth were eliminated in the presence of CI-1033.

CONCLUSIONS

These findings identify the ErbB signaling network as a target for GLP-<em>2</em> action leading to stimulation of growth factor-dependent signal transduction and bowel growth in vivo.

EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming <em>growth</em> <em>factor</em>-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal <em>keratinocyte</em> layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/<em>2</em> pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/<em>2</em> phosphorylation that in turn inhibited Smad<em>2</em> signaling by phosphorylation of residues Ser<em>2</em>45/<em>2</em>50/<em>2</em>55. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for <em>keratinocyte</em> α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.

<em>Growth</em> inhibitor/type beta transforming <em>growth</em> <em>factor</em> purified from BSC-1 cells and human platelets is shown to strongly inhibit the proliferation of Con A-stimulated mouse thymocytes. The inhibition can be achieved with <em>growth</em> inhibitor/type beta transforming <em>growth</em> <em>factor</em> concentrations approximately equal to 1/10th those necessary to inhibit <em>keratinocyte</em> cultures. The inhibitory effect in thymocyte cultures can be reversed by the addition of interleukin <em>2</em>. These findings suggest that <em>growth</em> inhibitor/type beta transforming <em>growth</em> <em>factor</em> is a naturally occurring immunoregulator.

Transforming <em>Growth</em> <em>Factor</em> (TGF-beta1) is a member of the TGF-beta superfamily ligand-receptor network. and plays a crucial role in tissue regeneration. The extensive in vitro and in vivo experimental literature describing its actions nevertheless describe an apparent paradox in that during re-epithelialisation it acts as proliferation inhibitor for <em>keratinocytes</em>. The majority of biological models focus on certain aspects of TGF-beta1 behaviour and no one model provides a comprehensive story of this regulatory factor's action. Accordingly our aim was to develop a computational model to act as a complementary approach to improve our understanding of TGF-beta1. In our previous study, an agent-based model of <em>keratinocyte</em> colony formation in <em>2</em>D culture was developed. In this study this model was extensively developed into a three dimensional multiscale model of the human epidermis which is comprised of three interacting and integrated layers: (1) an agent-based model which captures the biological rules governing the cells in the human epidermis at the cellular level and includes the rules for injury induced emergent behaviours, (<em>2</em>) a COmplex PAthway SImulator (COPASI) model which simulates the expression and signalling of TGF-beta1 at the sub-cellular level and (3) a mechanical layer embodied by a numerical physical solver responsible for resolving the forces exerted between cells at the multi-cellular level. The integrated model was initially validated by using it to grow a piece of virtual epidermis in 3D and comparing the in virtuo simulations of <em>keratinocyte</em> behaviour and of TGF-beta1 signalling with the extensive research literature describing this key regulatory protein. This research reinforces the idea that computational modelling can be an effective additional tool to aid our understanding of complex systems. In the accompanying paper the model is used to explore hypotheses of the functions of TGF-beta1 at the cellular and subcellular level on different <em>keratinocyte</em> populations during epidermal wound healing.

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte <em>growth</em> <em>factor</em> activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human <em>keratinocytes</em> are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human <em>keratinocytes</em> when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and <em>2</em>) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Epilysin (MMP-<em>2</em>8) is the newest member of the matrix metalloproteinase enzyme family. Several members of this enzyme family have been associated with various aspects of wound repair and cancer invasion. The aim of this study was to characterize in different types of wounds, skin cancers, and <em>keratinocyte</em> cultures <em>factors</em> that contribute to epilysin expression in vivo, as well as how and where it is induced in relation to other matrix metalloproteinases. Our results indicate that epilysin is produced by the mitotic Ki-67-positive <em>keratinocytes</em> distal from the wound edge in both acute and chronic wounds and that it does not generally colocalize with collagenase-1, stromelysin-<em>2</em>, or 9<em>2</em> kDa gelatinase in migrating <em>keratinocytes</em>. An injury of epidermis was needed for epilysin induction as it was upregulated in ulcerated pyogenic granulomas and in suction blisters but was not detected in intact acanthotic or normal skin. Unlike many other matrix metalloproteinases, epilysin was not detected in the invading cancer cell nests of sclerosing basal or squamous cell cancers of various grades. When primary <em>keratinocytes</em> were stimulated with tumor necrosis <em>factor</em> alpha, upregulation of epilysin mRNA was evident within <em>2</em>4-48 h as measured by quantitative reverse transcription polymerase chain reaction. In primary <em>keratinocyte</em>, HaCaT, and A431 carcinoma cell cultures none of the 10 other <em>growth</em> <em>factors</em> or extracellular matrices studied were able to upregulate epilysin expression. Our results suggest that epilysin expression is tightly spatially and temporally regulated during wound repair. Although the in vivo substrates of epilysin are not known at present, its expression pattern suggests that it may be needed to restructure the basement membrane or to degrade adhesive proteins between <em>keratinocytes</em> to supply new cells for the migrating front.

Fibroblast <em>growth</em> <em>factors</em> (FGFs) require heparan sulfate proteoglycans (HSPGs) as co<em>factors</em> for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-<em>2</em> binds to <em>keratinocytes</em> and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-<em>2</em>, FR1-AP binds avidly to FGF-<em>2</em> immobilized on <em>keratinocyte</em> cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-<em>2</em>. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.

Binding of the zymogen serine protease <em>Factor</em> VII (FVII) to its cellular co<em>factor</em> tissue <em>factor</em> (TF) triggers blood coagulation. Several recent reports have suggested that the formation of this complex may serve additional functions. We have used cDNA arrays to study differential gene expression in response to the interaction of activated FVII (FVIIa) with TF on a human <em>keratinocyte</em> cell line. Of 931 mRNA species observed up to 6 h after FVIIa (10 nM) addition, <em>2</em>4 were significantly up-regulated in what may resemble a wound-type response. Responders included mRNA species coding for transcription regulators (c-fos, egr-1, ETR101, BTEB<em>2</em>, c-myc, fra-1, and tristetraproline), <em>growth</em> <em>factors</em> (amphiregulin, hbEGF, CTGF, and FGF-5), proinflammatory cytokines (IL-1beta, IL-8, LIF, and MIP<em>2</em>alpha), proteins involved in cellular reorganization/migration (RhoE, uPAR, and collagenases 1 and 3), and others (PAI-<em>2</em>, cyclophilin, GADD45, Jagged1, and prostaglandin E(<em>2</em>) receptor). The transcriptional response to FVIIa was abrogated by antibodies to TF and left unaffected by hirudin. The pattern of genes induced suggests that the FVIIa.TF complex may play an active role in early wound repair as well as hemostasis. The former is a novel function ascribed to the complex that may also be contributing to the pathophysiology of unwarranted TF expression.

Up regulation of matrix metalloproteinases (MMPs), particularly collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and gelatinase A (MMP-<em>2</em>) is responsible for the lysis of dermal collagen and elastin fibers during chronological skin aging. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural MMP inhibitor family, encompassing four members. Its expression is decreased with fibroblast senescence, both ex vivo and in vivo, thus contributing to increased catabolic activity within dermis. TIMP-1 displays multiple biological functions. It inhibits most MMPs, except membrane-type MMP subfamily, with Ki in the subnanomolar range, but also interacts with the hemopexin-like (PEX) domain of pro MMP-9. Besides, it exhibits <em>keratinocyte</em> and fibroblast <em>growth</em> <em>factor</em>-like activity and has been described as a cell survival <em>factor</em>.

Many tumors show a mutant or inactive tumor suppressor p53 (TP53) status, and TP53 in the tumor-carrying human papillomavirus (HPV) may be dysfunctional because of inhibition by the viral protein HPV E6. Molecular mechanisms underlying radiation responses and the radiation-induced resistant phenotype in the TP53-inactive tumor have not been well investigated. In the present study, using a human <em>keratinocyte</em> line (HK18) with TP53 inhibited by HPV18 infection, we demonstrated that nuclear <em>factor</em> (NF)-kappaB is responsible for a major portion of the radioresistance observed in a cell population (HK18-IR) derived from HK18 cells by fractionated ionizing radiation (FIR; <em>2</em> Gy/fraction; total dose, 60 Gy). HK18-IR cells showed increased clonogenic radioresistance [dose-modifying <em>factor</em> (DMF), 1.47], reduced apoptotic response, and a shortened radiation-induced <em>growth</em> delay. Both DNA-binding and reporter transcriptional activity of NF-kappaB, but not of TP53, were activated in HK18-IR cells compared with the parental HK18 cells; this activation was observed both before and after a single dose of 5 Gy. To determine target genes responsive to NF-kappaB activation, DNA microarray profiles for 588 genes were matched in HK18-IR cells compared with those in HK18 cells; the paired comparisons were made for basal levels before irradiation or for levels <em>2</em>4 h after 5 Gy. For <em>2</em>5 genes, a <em>2</em>- to 5-fold up-regulation in HK18-IR cells relative to HK18 cells was similar when comparisons were made for basal levels or for levels after irradiation. Included in the approximately 4% of genes activated in HK18-IR cells, were six genes (Cyclin B1, Cyclin D1, HIAP, BAG-1, TTF, and fibronectin) putatively linked to NF-kappaB regulation. We then measured the expression of this group of FIR-regulated genes in HK18-IR cells expressing a dominant-negative mutant IkappaB (mIkappaB) that inhibited NF-kappaB activation. Clonogenic radioresistance was reduced greatly in the mIkappaB transfectants (DMF, 1.18 and 1.10, respectively, at 10% and 1% of isosurvival for mIkappaB transfectants compared with 1.47 and 1.45, respectively, for vector control transfectants). Expressions of Cyclin B1, Cyclin D1, and HIAP were down-regulated by the inhibition of NF-kappaB. These results suggest that transcription of NF-kappaB and a group of NF-kappaB target genes are involved in radioresistance in FIR-treated tumor cells with inactive TP53.

We previously revealed a novel signal pathway involving S100A11 for inhibition of the <em>growth</em> of normal human <em>keratinocytes</em> (NHK) caused by high Ca(++) or transforming <em>growth</em> <em>factor</em> beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p<em>2</em>1(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; <em>2</em>) extracellular S100A11 acts on NHK to enhance the production of epidermal <em>growth</em> <em>factor</em> family proteins, resulting in <em>growth</em> stimulation; 3) receptor for advanced glycation end products, nuclear <em>factor</em>-kappaB, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in <em>growth</em> regulation of epithelial cells.

In vitro generated skin models find <em>growing</em> interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. <em>Growth</em> conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured <em>keratinocytes</em>. These SEs demonstrate sustained epidermal viability - over 1<em>2</em> weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured <em>keratinocytes</em> exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast <em>growth</em> <em>factors</em>. Consequently, precultivation of dermal equivalents (DEs) in basal or <em>growth</em> <em>factor</em>-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4)cells/cm(<em>2</em>) on epidermal tissue quality equalled that of the standard density (<em>2</em> x 10(5)cells/cm(<em>2</em>)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to <em>2</em> days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual <em>keratinocyte</em>-fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.

Transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) regulates the tissue response to injury and is the principal driver of excessive scarring leading to fibrosis and eventual organ failure. The TGF-β1 effectors SMAD3 and p53 are major contributors to disease progression. While SMAD3 is an established pro-fibrotic <em>factor</em>, the role of p53 in the TGF-β1-induced fibrotic program is not clear. p53 gene silencing, genetic ablation/subsequent rescue, and pharmacological inhibition confirmed that p53 was required for expression of plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1 target gene and a key causative element in fibrotic disorders. TGF-β1 regulated p53 activity by stimulating p53(Ser15 and 9) phosphorylation and acetylation, promoting interactions with activated SMADs and subsequent binding of p53/SMAD3 to the PAI-1 promoter in HK-<em>2</em> human renal tubular epithelial cells and HaCaT human <em>keratinocytes</em>. Immunohistochemistry revealed prominent co-induction of SMAD3, p53 and PAI-1 in the tubular epithelium of the obstructed kidney consistent with a potential in vivo role for p53 and SMADs in TGF-β1-driven renal fibrosis. TGF-β1-initiated phosphorylation of p53(Ser15) and up-regulation of expression of several pro-fibrotic genes, moreover, was dependent on the rapid generation of reactive oxygen species (ROS). shRNA silencing of the p<em>2</em><em>2</em>(Phox) subunit of NADP(H) oxidases in HK-<em>2</em> cells partially attenuated (over 50%) p53(Ser15) phosphorylation and PAI-1 induction. These studies highlight the role of free radicals in p53 activation and subsequent pro-fibrotic reprogramming by TGF-β1 via the SMAD3-p53 transcriptional axis. Present findings provide a rationale for therapeutic targeting of SMAD3-p53 in aberrant TGF-β1 signaling associated with renal fibrosis.

Wound healing, including re-epithelialization, is delayed in diabetes. <em>Growth</em> <em>factors</em> influence the healing process and amongst these, insulin-like <em>growth</em> <em>factor</em> (IGF) has been shown to stimulate <em>keratinocyte</em> proliferation in vitro. Monoclonal antibodies to insulin-like <em>growth</em> <em>factors</em> 1 and <em>2</em> (IGF1 and IGF<em>2</em>) were used to investigate their distribution in diabetic foot ulcers and surrounding tissues by immunohistochemistry, compared with diabetic and non-diabetic uninjured skin. IGF<em>2</em> was found throughout the epidermis (stratum granulosum, spinosum, and basale) in all three groups. Staining for IGF<em>2</em> was intense in both normal and diabetic skin as well as in diabetic foot ulcers, being greatest at the ulcer edge. IGF1, in comparison, was found throughout the epidermis of non-diabetic skin; expression was restricted to the stratum granulosum and spinosum of uninjured diabetic skin and was absent in the basal layer at the ulcer edge. A similar absence of IGF1 in dermal fibroblasts was found in tissue sections from diabetic patients. This lack of expression of IGF1 within the basal layer and fibroblasts may contribute to retarded wound healing in diabetes mellitus.

Interleukin-17 (IL-17) is a proinflammatory cytokine mainly produced by activated CD4+ CD45RO T cells. In mice, we have demonstrated that, depending on the model, IL-17 may act as a tumor <em>growth</em>-promoting or -inhibiting <em>factor</em>. In order to address the relevance of these models in human tumors, we look for the natural expression and activity of IL-17 in mycosis fungoides (MF) and Sezary syndrome (SS). These cutaneous T-cell lymphomas were selected because they are usually CD3+ CD4+ CD45RO+, a phenotype similar to nontransformed T cells producing IL-17. We show that in vitro activated malignant T cells derived from MF or SS patients express IL-17 mRNA and secrete this cytokine. However, IL-17 does not act in vitro as a <em>growth</em> <em>factor</em> for MF or SS cell lines. In addition, 5 out of 10 MF/SS biopsies expressed IL-17 mRNA, while this cytokine was not detected in normal skin. IL-17 was not observed in the biopsies derived from <em>2</em> patients initially identified as MF, whereas an upregulation of this cytokine was clearly demonstrated during progression of the disease in these patients. An association between IL-17 expression and polymorphonuclear neutrophil infiltration was also recorded in this group of MF/SS patients. A more detailed analysis of <em>2</em> patients with a pustular form of MF and SS revealed that IL-17 may participate in the recruitment of polymorphonuclear neutrophils via a paracrine mechanism involving <em>keratinocyte</em>-released IL-8. This study is the first report demonstrating that some human tumor cells could express IL-17, a cytokine that represents an early event in the development of the inflammatory reaction within the tumor microenvironment, a process that may influence tumor phenotype and <em>growth</em>.

The proteolytic processing of laminin-5 at the short arm of the gamma<em>2</em> chain (gamma<em>2</em>sa) is known to convert this laminin from a cell adhesion type to a motility type. Here, we studied this mechanism by analyzing the functions of gamma<em>2</em>sa. In some immortalized or tumorigenic human cell lines, a recombinant gamma<em>2</em>sa, in either soluble or insoluble (coated) form, promoted the adhesion of these cells to the processed laminin-5 (Pr-LN5), and it suppressed their migration stimulated by serum or epidermal <em>growth</em> <em>factor</em> (EGF). Gamma<em>2</em>sa also suppressed EGF-induced tyrosine phosphorylation of integrin beta4 and resultant disruption of hemidesmosome-like structures in <em>keratinocytes</em>. Gamma<em>2</em>sa bound to syndecan-1, and this binding, as well as its cell adhesion activity, was blocked by heparin. By analyzing the activities of three different gamma<em>2</em>sa fragments, the active site of gamma<em>2</em>sa was localized to the NH(<em>2</em>)-terminal EGF-like sequence (domain V or LEa). Suppression of syndecan-1 expression by the RNA interference effectively blocked the activities of domain V capable of promoting cell adhesion and inhibiting the integrin beta4 phosphorylation. These results demonstrate that domain V of the gamma<em>2</em> chain negatively regulates the integrin beta4 phosphorylation, probably through a syndecan-1-mediated signaling, leading to enhanced cell adhesion and suppressed cell motility.

The proto-oncogene c-myc is involved in the regulation of cell proliferation, differentiation, and apoptosis. In this study, we used an inducible transgenic mouse model in which c-Myc was targeted to the epidermis and, after activation, gave rise to hyperplastic and dysplastic skin lesions and to dermal angiogenesis, involving both vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor-1 and VEGF receptor-<em>2</em>. After c-Myc activation, VEGF mRNA was expressed in postmitotic <em>keratinocytes</em> where it colocalized with transgene expression and areas of tissue hypoxia, suggesting a role of hypoxia in VEGF induction. In vitro, c-Myc activation alone was able to induce VEGF protein release and in conjunction with hypoxia, c-Myc activation further increased VEGF protein. Blocking VEGF signaling in vivo significantly reduced dermal angiogenesis, demonstrating the importance of VEGF as a mediating <em>factor</em> for the c-Myc-induced angiogenic phenotype.

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal <em>keratinocytes</em> of healthy human skin. The human <em>keratinocyte</em> cell line HaCaT, which like basal <em>keratinocytes</em> constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like <em>growth</em> <em>factor</em> (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-<em>2</em> or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal <em>keratinocytes</em>.

The metalloproteinase ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) is induced under inflammatory conditions, and it is also a potent inhibitor of angiogenesis. Due to these properties, we speculated about the role of ADAMTS1 in cutaneous wound repair. Here we have shown up-regulation of ADAMTS1 expression in wounds of normal and particularly of healing-impaired genetically diabetic mice. Immunofluorescence staining identified macrophages as the source of ADAMTS1 in early wounds, whereas <em>keratinocytes</em> and fibroblasts produce this protein at later stages of wound healing. The distribution of ADAMTS1 in the normal and wounded epidermis, its regulation in cultured <em>keratinocytes</em>, as well as the skin phenotype of ADAMTS1 knock-out mice suggests a role of this metalloproteinase in <em>keratinocyte</em> differentiation. Furthermore, we provide evidence for a novel dual function of ADAMTS1 in fibroblast migration; although low concentrations of this protein stimulate fibroblast migration via its proteolytic activity, high concentrations inhibit this process because of binding to fibroblast <em>growth</em> <em>factor</em>-<em>2</em> and subsequent inhibition of its promotogenic activity. Similar effects were also observed with endothelial cells. Taken together, our results suggest a role of ADAMTS1 in <em>keratinocyte</em> differentiation and migration of fibroblasts and endothelial cells in healing skin wounds.

Efficient wound repair is essential for the maintenance of the integrity of the skin. The repair process is controlled by a variety of <em>growth</em> <em>factors</em> and cytokines, and their abnormal expression or activity can cause healing disorders. Here, we show that wound repair is severely delayed in mice lacking fibroblast <em>growth</em> <em>factor</em> receptors (FGFR) 1 and <em>2</em> in <em>keratinocytes</em>. As the underlying mechanism, we identified impaired wound contraction and a delay in re-epithelialization that resulted from impaired <em>keratinocyte</em> migration at the wound edge. Scratch wounding and transwell assays demonstrated that FGFR1/<em>2</em>-deficient <em>keratinocytes</em> had a reduced migration velocity and impaired directional persistence owing to inefficient formation and turnover of focal adhesions. Underlying this defect, we identified a significant reduction in the expression of major focal adhesion components in the absence of FGFR signaling, resulting in a general migratory deficiency. These results identify FGFs as key regulators of <em>keratinocyte</em> migration in wounded skin.

The migration of human <em>keratinocytes</em> across the would bed is an early and critical event in the reepithelialization of cutaneous wounds. Epidermal <em>growth</em> <em>factor</em> (EGF) has been shown to accelerate the healing of fresh, split-thickness cutaneous wounds when applied topically. The mechanism(s) by which this accelerated healing occurs remains unknown. Using an assay that directly evaluates human <em>keratinocyte</em> locomotion without confounding the possibility of cell proliferation, we examined the influence of EGF on human <em>keratinocyte</em> motility. Both recombinant epidermal <em>growth</em> <em>factor</em> (rEGF) and transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) promoted human <em>keratinocyte</em> locomotion when the cells were apposed to connective tissue matrices of collagen or fibronectin, important components of the wound bed. Other <em>growth</em> <em>factors</em> studied did not enhance <em>keratinocyte</em> migration. Blocking the EGF/TGF-alpha receptor on the cell surface of <em>keratinocytes</em> with specific antibody inhibited the stimulation of <em>keratinocyte</em> locomotion by rEGF and TGF-alpha. Flow cytometry analysis of <em>keratinocytes</em> migrating on type I collagen in the presence of rEGF or TGF-alpha revealed increased expression of the alpha <em>2</em> integrin subunit on the <em>keratinocyte</em> surface. The alpha <em>2</em> beta 1 integrin mediates <em>keratinocyte</em> migration on collagens type I and IV, and inhibition of migration via antibody blockade of the alpha <em>2</em> beta 1 integrin can be partially overcome by increasing the concentration of rEGF present in the medium. Our study demonstrates that the <em>growth</em>-independent stimulation of <em>keratinocyte</em> locomotion via regulation of integrin expression may be one mechanism by which EGF accelerates the reepithelialization of human cutaneous wounds.