Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for lung cancer and an aberrant microbiota of the lung. Microbial colonization contributes to chronic neutrophilic inflammation in COPD. Nontypeable Haemophilus influenzae (NTHi) is frequently found in lungs of stable COPD patients and is the major pathogen triggering exacerbations. The epithelial cytokine interleukin-17C (IL-17C) promotes the recruitment of neutrophils into inflamed tissues. The purpose of this study was to investigate the function of IL-17C in the pulmonary tumor microenvironment. We subjected mice deficient for IL-17C (IL-17C-/-) and mice double deficient for Toll-like receptor <em>2</em> and 4 (TLR-<em>2</em>/4-/-) to a metastatic lung cancer model. Tumor proliferation and <em>growth</em> as well as the number of tumor-associated neutrophils was significantly decreased in IL-17C-/- and TLR-<em>2</em>/4-/- mice exposed to NTHi. The NTHi-induced pulmonary expression of IL-17C was dependent on TLR-<em>2</em>/4. In vitro, IL-17C increased the NTHi- and tumor necrosis <em>factor</em>-α-induced expression of the neutrophil chemokines <em>keratinocyte</em>-derived chemokine and macrophage inflammatory protein <em>2</em> in lung cancer cells but did not affect proliferation. Human lung cancer samples stained positive for IL-17C, and in non-small cell lung cancer patients with lymph node metastasis, IL-17C was identified as a negative prognostic <em>factor</em>. Our data indicate that epithelial IL-17C promotes neutrophilic inflammation in the tumor microenvironment and suggest that IL-17C links a pathologic microbiota, as present in COPD patients, with enhanced tumor <em>growth</em>.

BACKGROUND

Over the last decade, the incidence of ultraviolet B (UVB)-related skin problems has increased. Oxidative stress caused by UVB induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyperpigmentation. Therefore, increasing the antioxidant abilities of skin cells is thought to be a beneficial strategy for the development of sunscreen agents. Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that is known to exhibit antioxidant properties.

OBJECTIVE

The purpose of this study was to investigate the effect of SOD1 on alpha-melanocyte stimulating hormone (α-MSH) and UVB-induced melanogenesis in B16F10 melanoma cells and HRM-2 melanin-possessing hairless mice.

METHODS

The inhibitory effect of SOD1 on tyrosinase activity was evaluated in a cell-free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of SOD1 in vitro, and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of SOD1 in vivo.

RESULTS

We found that SOD1 inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 melanoma cells. SOD1 did not inhibit tyrosinase activity under cell-free conditions. The results indicate that SOD1 may reduce pigmentation by an indirect, nonenzymatic mechanism. We also found that SOD1 decreased UVB-induced melanogenesis in HRM-2 melanin-possessing hairless mice, as visualized through hematoxylin and eosin staining and Fontana-Masson staining.

CONCLUSIONS

Our results indicate that SOD1 has an inhibitory effect on α-MSH and UVB-induced melanogenesis, indicating that SOD1 may be a promising sunscreen agent.

Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast <em>Growth</em> <em>Factors</em> (FGFs) and their receptors FGFRs are recognized as key elements. FGFR<em>2</em> gene encodes for two splicing transcript variants, FGFR<em>2</em>-IIIb or <em>Keratinocyte</em> <em>Growth</em> <em>Factor</em> Receptor (KGFR) and FGFR<em>2</em>-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR<em>2</em> mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis <em>Factor</em>-α, Interleukin-1β and Interleukin <em>2</em>. Furthermore, we found that TNFα determines FGFR<em>2</em> transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription <em>factor</em> E<em>2</em>F1. Experiments based on FGFR<em>2</em> promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E<em>2</em>F1. Computational analysis indicated that this element is a non-canonical E<em>2</em>F responsive motif. Thus far, the molecular mechanisms of FGFR<em>2</em> upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR<em>2</em> expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR<em>2</em> expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

Although initially thought to improve an individual's ability to heal, mechanical unloading promoted by extended periods of bed rest has emerged as a contributing <em>factor</em> to delayed or aberrant tissue repair. Using a rat hindlimb unloading (HLU) model of hypogravity, we mimicked some aspects of physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. This model simulates bed rest in that the animal undergoes physiological adaptations, resulting in a reduction in exercise capability, increased frequency of orthostatic intolerance, and a reduction in plasma volume. To investigate whether changes associated with prior prolonged bed rest correlate with impaired cutaneous wound healing, we examined wound closure, angiogenesis, and collagen content in day <em>2</em> to day <em>2</em>1 wounds from rats exposed to HLU <em>2</em> wk before excisional wounding. Wound closure was delayed in day <em>2</em> wounds from HLU rats compared with ambulatory controls. Although the levels of proangiogenic <em>growth</em> <em>factors</em>, fibroblast <em>growth</em> <em>factor</em>-<em>2</em> (FGF-<em>2</em>), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) were similar between the two groups, wound vascularity was significantly reduced in day 7 wounds from HLU animals. To further examine this disparity, total collagen content was assessed but found to be similar between the two groups. Taken together, these results suggest that <em>keratinocyte</em> and endothelial cell function may be impaired during the wound healing process under periods of prolonged inactivity or bed rest.

BACKGROUND

A predominantly T-helper type <em>2</em> (Th<em>2</em>) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota.

METHODS

We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice.

RESULTS

pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate.

CONCLUSIONS

These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal <em>keratinocyte</em> microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from <em>keratinocyte</em>-like cells (HaCaT) were added to fibroblast cultures, expression of <em>2</em>1 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming <em>growth</em> <em>factor</em>-β signaling. Similar changes were observed at the protein level. MVs from normal epidermal <em>keratinocytes</em> showed similar response to HaCaT cells. KC-MVs activated ERK1/<em>2</em>, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/<em>2</em> signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that <em>keratinocyte</em> microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing.

Fibroblast activation protein-α (FAP-α) promotes tumor <em>growth</em> and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk <em>factor</em> for malignant melanoma, influences the expression of FAP-α is unknown. We examined the effect of UVR on FAP-α expression in melanocytes, <em>keratinocytes</em> and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-α in fibroblasts, melanocytes and primary melanoma cells (PM) whereas <em>keratinocytes</em> and metastatic melanoma cells remained FAP-α negative. UVA and UVB stimulated FAP-α-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-α in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-α in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-α expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-β1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-α/DPPIV inhibitor Gly-ProP(OPh)<em>2</em> significantly decreased this response implicating FAP-α/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-α expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-α expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-β1 and Wnt5a and these <em>factors</em> should be evaluated as targets to reduce FAP-α activity and prevent early melanoma dissemination.

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. <em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), a potent epithelial cell mitogen, may have a role in preventing ventilator-induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF-<em>2</em> in VILI in rats. Forty healthy adult male Sprague-Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high-volume zero positive end-expiratory pressure) and HVZP+KGF-<em>2</em> were given intratracheally equal PBS and 5 mg/kg KGF-<em>2</em> 7<em>2</em> hrs before 4 hrs HVZP ventilation (<em>2</em>0 ml/kg), respectively, while PBS and KGF-<em>2</em> were administered in the same manner in Groups Control and KGF-<em>2</em>, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis <em>factor</em>-α, macrophage inflammatory protein <em>2</em>), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre-treatment with KGF-<em>2</em> showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF-<em>2</em> might be considered a promising prevention for human VILI or other acute lung injury diseases.

BACKGROUND

Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS.

RESULTS

The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity.

CONCLUSIONS

This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

In inflammatory bowel disease (IBD), obesity is associated with worsening of the course of disease. Here, we examined the role of obesity in the development of colitis and studied mesenteric fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity with the trinitrobenzene sulfonic acid (TNBS) colitis mouse model to create groups with obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed by histology, myeloperoxidase assay, and cytokine mRNA expression by real-time PCR. Medium from human mesenteric fat and cultured preadipocytes was obtained from obese patients and those with IBD. Histological analysis showed inflammatory cell infiltrate and increased histological damage in the intestine and mesenteric fat of obese mice with colitis compared with all other groups. Obesity also increased the expression of proinflammatory cytokines including IL-1β, TNF-α, monocyte chemoattractant protein 1, and <em>keratinocyte</em>-derived chemokine, while it decreased the TNBS-induced increases in IL-<em>2</em> and IFN-γ in mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese patients and those with and IBD demonstrated differential release of adipokines and <em>growth</em> <em>factors</em> compared with controls. Fat-conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460 colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced colitis. In conclusion, obesity worsens the outcome of experimental colitis, and obesity- and IBD-associated changes in adipose tissue promote differential mediator release in mesenteric fat that modulates colonocyte responses and may affect the course of colitis. Our results also suggest an important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during colitis.

BACKGROUND

While it is well known that both exogenous and endogenous stimuli can trigger appearance of psoriatic lesions, the initial cellular and molecular events mediated by immunocompetent cells normally resident in prepsoriatic (PN) skin are not well understood. Moreover, it is unclear whether there are any fundamentally important differences in the innate immune response of normal healthy skin (NN skin) versus PN skin. Since acute tissue responses to stimuli involve both resident cells and immunocytes recruited rapidly from circulation, it is difficult to discern the contribution of endogenous cells normally present in skin.

OBJECTIVE

To solely characterize the reactivity of resident immunocytes using an experimental system.

METHODS

To probe the activation potential of resident immunocytes in NN (n = 18) and PN skin (n = 10), a short-term ex vivo organ culture system containing interleukin (IL)-<em>2</em> was established and characterized. To mimic exogenous or environmental trigger <em>factors</em>, bacteria-derived superantigens and lipopolysaccharide were added to the skin-explant assays, whereas endogenous trigger <em>factors</em> were investigated using heat shock proteins.

RESULTS

Using this skin-explant assay, both NN and PN skin gave rise to an expansion of various T-cell subsets, which could differentially produce various cytokines and a growth factor (keratinocyte growth factor), depending on the stimulus and source of skin. Bacterial superantigens were relatively potent inducers of interferon-gamma, and natural killer-T cells were observed proliferating from PN skin.

CONCLUSIONS

Despite relatively few T cells normally residing in either NN or PN skin, initiation of skin explants from both sets of individuals in the presence of IL-<em>2</em> triggered vigorous T-cell proliferation and cytokine/growth factor release. These results demonstrate the utility of this skin-explant assay system to further investigate quantitative and qualitative immune responses of NN and PN skin.

Generally, exposure to LPS in human airways occurs in the form of aerosols and causes an acute inflammatory response or exacerbates existing chronic inflammatory conditions by enhancing airway remodeling and associated pathologies. The present study evaluated which inflammatory mediators may be responsible for the expression of Bcl-<em>2</em> and mucus cell metaplasia when mice are exposed to aerosolized LPS. At 3 days after exposure, aerosolized LPS (for <em>2</em>0-40 min) with the estimated lung deposited dosage of 0, 0.0<em>2</em>, 0.<em>2</em>, 1.4, and <em>2</em>0.<em>2</em> μg showed a characteristic dose-dependent increase in polymorphonuclear neutrophils. Significant increases of proinflammatory mediators, including IL-1β, TNF-α, IL-6, <em>growth</em>-related oncogene or <em>keratinocyte</em>-derived cytokine, IFN-γ-induced protein-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α, were detected at the highest doses. In addition to increased numbers of airway epithelial cells, mucus cell numbers and mucus production were increased in a dose-dependent manner. Hyperplastic epithelial cells expressed insulin-like <em>growth</em> <em>factor</em> (IGF)-1 and, similar to previous studies, increased expression of the prosurvival protein Bcl-<em>2</em> and induced expression of Muc5ac. Suppression of IGF-1 expression using retroviral shRNA blocked Bcl-<em>2</em> expression in human and murine airway epithelial cells and Muc5ac in primary murine airway epithelial cells. These findings show that acute inflammation induces IGF-1 to mediate Bcl-<em>2</em> and Muc5ac expression in airway epithelial cells.

OBJECTIVE

To investigate the pathophysiology of radiation-induced wounds of the head and neck at a molecular level.

METHODS

Basic science, prospective study.

METHODS

The study was conducted at the Department of Otolaryngology-Head and Neck Surgery, Ruprecht Karls-University Heidelberg, Faculty of Medicine Mannheim, Mannheim, Germany.

METHODS

Keratinocytes from chronic nonhealing ulcers in irradiated areas as well as from healthy skin areas in the same patients (n = 3) were harvested during surgical procedures and isolated in cell culture. First, a proliferation assay was performed. Gene expression was analyzed by microarray, protein expression by immunohistochemistry.

RESULTS

Keratinocytes from radiogenic wounds showed a shift from the high molecular keratins 1 and 10 to the low molecular keratins 5 and 14 compared to normal control skin. Keratinocytes from nonhealing wounds showed a decreased expression of transforming growth factor alpha and beta 1, fibroblast growth factor 1 and 2, keratinocyte growth factor, vascular endothelial growth factor, and hepatocyte growth factor. The matrix metalloproteinases 2, 12, and 13 showed increased expression in irradiated keratinocytes and fibroblasts.

CONCLUSIONS

Our data showed a change of keratinocytes to a less differentiated state due to radiation. Additionally, it seems that radiation-induced dermal injuries often fail to heal because of decreased proliferation, impaired angiogenesis, and persistently high concentrations of matrix metalloproteinases.

We used a prematurely born rat/hyperoxia model of bronchopulmonary dysplasia (BPD) to test whether <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) treatment would protect against the development of several serious (cardio-)pulmonary complications of early life exposure to hyperoxia. KGF significantly protected against hyperoxic lethality (13-day survival rate = 50/64 (78%) for the O(<em>2</em>)-KGF vs. <em>2</em>9/66 (44%) for the O(<em>2</em>)-saline group, p < 0.001). Although KGF failed to protect against hyperoxic inhibition of normal postnatal alveoli formation and early pulmonary fibrosis, KGF consistently had a significant protective/preventive effect against the development of pulmonary hypertension during hyperoxia as reflected in comparative right ventricular hypertrophy: mean increase = +35% above normal for the O(<em>2</em>)-saline group vs. +3% for the O(<em>2</em>)-KGF premature rat group (p < 0.01).

<em>Growth</em> <em>factors</em> of the fibroblast <em>growth</em> <em>factor</em> (FGF) family bind receptors whose external domains are organized in a series of immunoglobulin-like loops. We engineered expression constructs in which cDNAs encoding individual immunoglobulin-like domains of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF/FGF-7) receptor were fused to the mouse immunoglobulin heavy chain Fc domain (HFc). Each chimera was efficiently secreted from NIH 3T3 transfectants and migrated at the predicted molecular mass after SDS/PAGE. Scatchard analysis revealed that the chimera containing immunoglobulin-like domains <em>2</em> (D<em>2</em>) and 3 (D3) bound KGF and acidic FGF at high affinities comparable to the native receptor. However, individual immunoglobulin-like domain chimeras demonstrated marked specificity in their ligand interactions. D<em>2</em>-HFc bound acidic FGF at high affinity, whereas it did not detectably interact with KGF. Conversely, D3-HFc bound KGF at high affinity but exhibited no detectable interaction with acidic FGF. Their selective ligand binding properties were confirmed by the specific neutralization of acidic FGF or KGF mitogenic activity using D<em>2</em> or D3 HFc, respectively. All of these findings establish that the major binding sites for related FGF ligands are localized to distinct receptor immunoglobulin-like domains.

Calorie restriction (CR) reduces the rate of cell proliferation in mitotic tissues. It has been suggested that this reduction in cell proliferation may mediate CR-induced increases in longevity. However, the mechanisms that lead to CR-induced reductions in cell proliferation rates remain unclear. To evaluate the CR-induced physiological adaptations that may mediate reductions in cell proliferation rates, we altered housing temperature and access to voluntary running wheels to determine the effects of food intake, energy expenditure, percent body fat, and body weight on proliferation rates of <em>keratinocytes</em>, liver cells, mammary epithelial cells, and splenic T-cells in C57BL/6 mice. We found that ∼<em>2</em>0% CR led to a reduction in cell proliferation rates in all cell types. However, lower cell proliferation rates were not observed with reductions in 1) food intake and energy expenditure in female mice housed at <em>2</em>7°C, <em>2</em>) percent body fat in female mice provided running wheels, or 3) body weight in male mice provided running wheels compared with ad libitum-fed controls. In contrast, reductions in insulin-like <em>growth</em> <em>factor</em> I were associated with decreased cell proliferation rates. Taken together, these data suggest that CR-induced reductions in food intake, energy expenditure, percent body fat, and body weight do not account for the reductions in global cell proliferation rates observed in CR. In addition, these data are consistent with the hypothesis that reduced cell proliferation rates could be useful as a biomarker of interventions that increase longevity.

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility <em>2</em> (ELMO<em>2</em>) as an ILK-binding protein in epidermal <em>keratinocytes</em>. Recently, we investigated the biological role of the ILK/ELMO<em>2</em> complexes, and found that they exist in the cytoplasm. ILK/ELMO<em>2</em> species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of <em>growth</em> <em>factors</em> and cytokines induce <em>keratinocyte</em> migration. However, we found that formation of RhoG/ELMO<em>2</em>/ILK complexes occurs selectively upon stimulation by epidermal <em>growth</em> <em>factor</em>, but not by transforming <em>growth</em> <em>factor</em>-β1 or <em>keratinocyte</em> <em>growth</em> <em>factor</em>. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.

In vivo, fibroblasts reside in connective tissues, with which they communicate in a reciprocal way. Such cell--extracellular matrix interactions can be studied in vitro by seeding fibroblasts in collagen lattices. Depending upon the mechanical properties of the system, fibroblasts are activated to assume defined phenotypes. In the present study, we examined a transcriptional profile of primary human dermal fibroblasts cultured in a relaxed collagen environment and found relative induction >><em>2</em>-fold) of 393 out of approx. 7100 transcripts when compared with the same system under mechanical tension. Despite down-regulated proliferation and matrix synthesis, cells did not become generally quiescent, since they induced transcription of numerous other genes including matrix metalloproteinases (MMPs) and <em>growth</em> <em>factors</em>/cytokines. Of particular interest was the induction of gene transcripts encoding pro-inflammatory mediators, e.g. cyclo-oxygenase-<em>2</em> (COX-<em>2</em>), and interleukins (ILs)-1 and -6. These are apparently regulated in a hierarchical fashion, since the addition of IL-1 receptor antagonist prevented induction of COX-<em>2</em>, IL-1 and IL-6, but not that of MMP-1 or <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). Our results suggest strongly that skin fibroblasts are versatile cells, which adapt to their extracellular environment by displaying specific phenotypes. One such phenotype, induced by a mechanically relaxed collagen environment, is the 'pro-inflammatory' fibroblast. We propose that fibroblasts that are embedded in a matrix environment can actively participate in the regulation of inflammatory processes.

Many experimental models have been proposed to study the pathophysiological features of emphysema, as well as to search for new therapeutic approaches for acute or chronically injured lung parenchyma. We aimed to characterize an emphysema model induced by multiple instillations of elastase by tracking changes in inflammation, remodeling, and cardiac function after each instillation. Forty-eight C57BL/6 mice were randomly assigned across two groups. Emphysema (ELA) animals received 1, <em>2</em>, 3, or 4 intratracheal instillations of pancreatic porcine elastase (PPE, 0.<em>2</em> IU) with a 1-week interval between them. Controls (C) received saline following the same protocol. Before and after implementation of the protocol, animals underwent echocardiographic analysis. After the first instillation of PPE, the percentage of mononuclear cells in the lung parenchyma increased compared to C (p = 0.0001). The second instillation resulted in hyperinflated alveoli, increased mean linear intercept, and reduced elastic fiber content in lung parenchyma compared to C (p = 0.0197). Following the third instillation, neutrophils and collagen fiber content in alveolar septa and airways increased, whereas static lung elastance was reduced compared to C (p = 0.0094). After the fourth instillation, the percentage of M1 macrophages in lungs; levels of interleukin-1β (IL-1β), <em>keratinocyte</em>-derived chemokine, hepatocyte <em>growth</em> <em>factor</em> (HGF), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF); and collagen fiber content in the pulmonary vessel wall were increased compared to C (p = 0.0096). At this time point, pulmonary arterial hypertension was apparent, with increased diastolic right ventricular wall thickness. In conclusion, the initial phase of emphysema was characterized by lung inflammation with predominance of mononuclear cells, whereas at the late stage, impairment of pulmonary and cardiovascular functions was observed. This model enables analysis of therapies at different time points during controlled progression of emphysema. Accordingly, early interventions could focus on the inflammatory process, while late interventions should focus on restoring cardiorespiratory function.

Mitogen-activated protein kinase kinases (MEK) 1 and <em>2</em> have crucial roles in tumorigenesis, cell proliferation, and protection from apoptosis, and their inhibition is therefore an attractive therapeutic strategy in cancer. Orally available and highly selective MEK inhibitors have been developed and assessed in numerous clinical trials, either alone or in combination with cytotoxic chemotherapy and/or other targeted agents. Of note, a complex picture of class-specific adverse effects associates with these drugs, frequently including inflammatory skin rash. Here, we investigated the response of normal human <em>keratinocytes</em> to the MEK inhibitors trametinib and cobimetinib, alone and in combination with the v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitors dabrafenib and vemurafenib, in terms of signal transduction and de novo gene expression. MEK inhibitors triggered enhanced expression of interferon regulatory <em>factor</em> 1 (IRF1) and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and up-regulated the <em>keratinocyte</em>-specific type I interferon κ (IFN-κ), the anti-viral effectors interferon-induced tetratricopeptide repeats (IFIT) 1 and <em>2</em>, and the pro-inflammatory chemokine (C-C motif) ligand <em>2</em> (CCL<em>2</em>) and the C-X-C motif chemokine 10 (CXCL10), both at the mRNA and protein level. Impairment of IRF1 expression, or abrogation of STAT1 phosphorylation due to IFN-κ gene silencing, suppressed anti-viral and pro-inflammatory gene expression. These data suggest that, similar to what we observed for epidermal <em>growth</em> <em>factor</em> receptor (EGFR) blockade, MEK inhibition activates a type I interferon response, which is now recognized as an effective anti-cancer response, in human epidermal <em>keratinocytes</em>.

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal <em>keratinocytes</em> and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified <em>keratinocytes</em> and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the <em>keratinocyte</em> sheet transiently increased and then decreased to two cell layers at <em>2</em> weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the <em>keratinocytes</em> and of <em>keratinocyte</em> <em>growth</em> <em>factor</em> 1 and <em>2</em> in the fibroblasts correlated with <em>keratinocyte</em> stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.

Vitamin D3 and its analogs are potent regulators of <em>growth</em> and differentiation of various cell types. A mechanism of action of vitamin D3 and other steroid hormones is to enhance the secretion of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) in target cells. In epidermal <em>keratinocytes</em>, vitamin D3 induced the expression of both TGF-beta 1 and TGF-beta <em>2</em> with minor changes in mRNA levels, while in BT-<em>2</em>0 breast carcinoma cells the increase in TGF-beta activity was preceded by an induction of mRNA. In both cell systems, the absolute amounts of active TGF-beta increased, and in <em>keratinocytes</em> the proportion of active TGF-beta was also enhanced. A concomitant enhancement of secretion of the latent TGF-beta-binding protein by vitamin D3 was observed in BT-<em>2</em>0 cells. Retinoic acid, which is known to interfere with vitamin D3 signaling, slightly decreased the levels of secreted TGF-beta 1 protein in BT-<em>2</em>0 cells, but did not significantly affect the vitamin D3-induced increase. In addition to regulation of the TGF-beta system, vitamin D3 decreases pericellular plasminogen activator activity in <em>keratinocytes</em>. Plasmin-mediated proteolytic events are involved in the release from pericellular space and activation of TGF-beta. We analyzed vitamin D3 regulation of fibroblast <em>growth</em> and the secretion of PA activity. Vitamin D3 inhibited fibroblast <em>growth</em> in a concentration-dependent manner and downregulated plasminogen activator activity as in <em>keratinocytes</em>. In fibroblasts, vitamin D3 did not induce notable alterations in TGF-beta 1 or latent TGF-beta-binding protein secretion, suggesting divergent <em>growth</em> inhibitory mechanisms. Our results indicate that vitamin D3 and its analogs are potent regulators of the TGF-beta and plasminogen activator systems in cells of epithelial and mesenchymal origin.

BACKGROUND

Autologous dermal fibroblasts may be useful in the treatment of skin wounds and for the enhancement of <em>keratinocyte</em> proliferation. This paper addressed the following questions: (1) can cultured fibroblasts (CF) be transplanted as suspensions to full-thickness skin wounds and do they influence wound healing; (<em>2</em>) will the transplanted CF be integrated into the new dermis; (3) can a transgene that encodes a secretable marker, human epidermal <em>growth</em> <em>factor</em> (hEGF), be expressed in the wound fluid by the transplanted CF; and (4) do CF cotransplanted with cultured <em>keratinocytes</em> (CK) influence the rate of wound healing?

METHODS

Suspensions of CF were transplanted alone or together with CK to full-thickness wounds covered with liquid-containing chambers in an established porcine model.

RESULTS

Transplantation of CF accelerated reepithelialization as determined from wound histologies and sequential measurements of protein efflux over the wound surface. CF transfected with a marker gene, beta-galactosidase, resulted in in vivo gene expression and demonstrated that transplanted CF integrated into the developing dermis. Transplantation of hEGF gene-transfected CF resulted in significant hEGF expression in wound fluid. The hEGF levels peaked at day 1 (<em>2</em>450 pg/ml) and then sharply decreased to low levels on day 6. CF cotransplanted with CK led to greater number of <em>keratinocyte</em> colonies in the wound and accelerated reepithelialization as compared with CK alone.

CONCLUSIONS

Transplanted CF integrated into the dermis, accelerated reepithelialization, and improved the outcome of CK transplantation. CF may also be used for the expression of transgenes in wound and wound fluid.

Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin alpha3A (lama3A) gene. To identify the TGF-beta-responsive elements (TGFbeta-REs) on the lama3A promoter, we have generated a series of 5'-deletions of the promoter upstream of the beta-galactosidase reporter gene. Transient cell transfection assays using mouse PAM<em>2</em>1<em>2</em> <em>keratinocytes</em> revealed that TGFbeta-REs lie between nucleotides -<em>2</em>97 and -54 relative to the transcription start site. Insertion of the TGFbeta-RE in front of the unresponsive minimal SV40 promoter conferred TGF-beta inducibility. Computer analysis of the promoter sequence identified three canonical activator protein-1 (AP-1) sites located at nucleotides -<em>2</em>77 (AP-1A), -1<em>2</em>5 (AP-1B), and -69 (AP-1C). Site-directed mutagenesis of either the AP-1A or AP-1C site did not drastically alter the basal activity of the lama3A promoter, but reduced TGF-beta responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-beta, suggesting a cooperative contribution of each site to the overall promoter activity. In contrast, mutation of the AP-1B site markedly reduced the basal activity of the lama3A promoter, indicating that this AP-1 site is essential for gene expression. Mobility shift assays demonstrated specific binding of Fra-<em>2</em> and JunD to the AP-1 sites, suggesting for the first time a possible regulatory function for the Fra-<em>2</em>.JunD AP-1 complex in a basal <em>keratinocyte</em>-specific gene.