<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-1 (KGF-1) is a member of the fibroblast <em>growth</em> <em>factor</em> (FGF) family FGF7 and is expressed in normal and wounded skin. KGF-1 is massively produced in the early stages of the wound healing process as well as during the later remodeling process (1, <em>2</em>). We have studied the effects of the electroporation of a KGF-1 plasmid into excisional wounds of different rodent models mimicking diseases known to impair the normal wound healing process. We have used a genetically diabetic mouse model and a septic rat model in our experiments, and we have shown improvement of the healing rate (9<em>2</em>% of the wounds are healed at day 1<em>2</em> vs. 40% of the control), the quality of epithelialization (histological score of 3.3 vs. 1.5), and the density of new blood vessels (85% more new blood vessels in the superficial layers than that of the control) (3, 4). Considering these results, we believe we can further explore the treatment modalities for using the electroporation-assisted transfection of DNA plasmid expression vectors of <em>growth</em> <em>factors</em> to enhance cutaneous wound healing.

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected <em>growth</em> <em>factors</em> (GF) on the attachment and <em>growth</em> of normal human <em>keratinocytes</em> in vitro. Single-cell suspensions of <em>keratinocytes</em> from near-confluent primary plates, plated on 5-10 microgram/cm<em>2</em> HFN, showed approximately 30-40% attachment after <em>2</em>-<em>2</em>4 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these <em>factors</em>. <em>Keratinocytes</em> grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-1<em>2</em> days at densities greater than or equal to 104 cell/cm<em>2</em>. Removal of most GF individually from the medium had little or no effect on <em>growth</em>, while removal of epidermal <em>growth</em> <em>factor</em> (EGF) alone reduced <em>growth</em> by 30-35% and removal of bovine brain extract (BE) alone reduced <em>growth</em> by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control <em>growth</em>, BE alone supported 30-40% control <em>growth</em>, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal <em>keratinocyte</em> morphology and protein production. These findings expand earlier observations that HFN facilitates <em>keratinocyte</em> attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured <em>keratinocytes</em>.

In the human <em>keratinocyte</em> cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal <em>growth</em> <em>factor</em> (EGF), bradykinin, thapsigargin, and the Ca(<em>2</em>+)-ionophore A<em>2</em>3187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A<em>2</em>3187 was sustained for at least 15 min. Extracellular Ca<em>2</em>+ and a rise in intracellular Ca<em>2</em>+ concentration ([Ca<em>2</em>+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca<em>2</em>+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca<em>2</em>+]i elevation of less than twofold, whereas with thapsigargin and A<em>2</em>3187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca<em>2</em>+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca<em>2</em>+]i generated by <em>growth</em> <em>factors</em> and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca<em>2</em>+]i has been linked to cell proliferation, differentiation, and apoptosis.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a fibroblast <em>growth</em> <em>factor</em> (FGF) family member that acts specifically on cells of epithelial origin. Its receptor (KGFR) is a membrane-spanning tyrosine kinase, which also binds acidic FGF (aFGF) with equally high affinity, and basic FGF (bFGF) with much lower affinity. The KGFR is encoded by the bek/FGFR-<em>2</em> gene, whose alternative transcript specifies a receptor with high affinity for aFGF and bFGF, but no detectable binding of KGF. The only structural difference between these two receptors is a 49-amino acid segment in the extracellular domain that is determined by single alternative exons. We report that a synthetic peptide (NH<em>2</em>-His199...Tyr<em>2</em><em>2</em>3-COOH) corresponding to part of the predicted sequence of the KGFR alternative exon blocks KGF mitogenic activity and the interaction between KGF and its receptor. The peptide also blocks the interaction between KGF and a neutralizing monoclonal antibody raised against this <em>growth</em> <em>factor</em>. These results demonstrate that the peptide binds directly and specifically to KGF and argue that this region of the receptor constitutes part or all of the KGF binding site.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is involved in the control of proliferation and differentiation of human <em>keratinocytes</em>. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>. We have previously shown (C. Marchese et al., Cell <em>Growth</em> Differ., 8: 989-997, 1997) that differentiation of primary cultured <em>keratinocytes</em> triggered by high Ca<em>2</em>+ concentrations in the <em>growing</em> medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human <em>keratinocyte</em> cell line HaCaT, widely used as a model to study <em>keratinocyte</em> differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

Mutation, deactivation and disregulated expression of oncogenes and tumour-suppressor genes may be involved in the pathogenesis of oral squamous cell carcinoma (SCC). Deactivation of the p53 tumour-suppressor gene allows cell proliferation and blocks apoptosis of malignant oral <em>keratinocytes</em>. Mutation in the ras oncogene results in persistent mitogenic signalling. Upregulatioed c-Myc expression, in the presence of <em>growth</em> <em>factors</em>, provides an additional proliferative signal. Loss of retinoblastoma tumour-suppressor gene (Rb) function may contribute to oral <em>keratinocyte</em> hyperproliferation and recent evidence suggests that simultaneous deactivation of both p53 and Rb is required for tumourigenesis. Enhanced Bcl-<em>2</em> and reduced Fas expression inhibit tumour cell apoptosis and may convey resistance to cytotoxic drugs and T cell-mediated cytotoxicity, respectively. Exogenous mutagens such as tobacco, alcohol and viral oncogenes may cause altered expression of oncogenes and tumour-suppressor genes in some cases of oral SCC. The impact of these mechanisms on future therapies for oral SCC is highlighted.

c-Src potentiates proliferation, survival, and invasiveness in response to epidermal <em>growth</em> <em>factor</em> (EGF) in human mammary carcinoma cells. Tyrosine (Tyr) 845 of ErbB1 is phosphorylated by Src and has been implicated in control of malignant behavior. Although several lines of evidence also suggest important interactions of ErbB and Src family kinase signaling in normal epithelial cells, little is known about the mechanism of this interaction. Studying normal human <em>keratinocytes</em> (NHKs), here we demonstrate strong expression of the Src family kinases Src, Yes, and Fyn; Src family kinase-dependent stimulation of Tyr 845 by EGF; and potent inhibition of NHK proliferation and migration by two Src family kinase inhibitors PP1 and PD17395<em>2</em>. EGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation occurred at much lower concentrations of EGF than required to phosphorylate Tyr 845. Moreover, the effect of Src family kinase inhibitors on EGF-stimulated ERK phosphorylation was transient, prompting a search for other targets of Src family kinase action. By enzyme-linked immunosorbent assay analysis, we found that three different Src family kinase inhibitors [6-(<em>2</em>,6-dichlorophenyl)-8-methyl-<em>2</em>-(4-morpholin-4-ylphenylamino)-8H-pyrido[<em>2</em>,3-d]pyrimidin-7-one (PD17395<em>2</em>), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and <em>2</em>-oxo-3-(4,5,6,7-tetrahydro-1H-indol-<em>2</em>-ylmethylene)-<em>2</em>,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (SU6656)] markedly inhibited elaboration of soluble amphiregulin by NHKs. The ErbB inhibitor PD158780 and the mitogen-activated protein kinase kinase inhibitor U01<em>2</em>6 also markedly inhibited NHK proliferation, migration, and amphiregulin production. Together, these observations demonstrate that one or more Src family kinases act upstream as well as downstream of ErbB1 to promote amphiregulin-dependent autocrine stimulation of NHKs and suggest that autocrine NHK proliferation is more dependent upon ERK activation than upon Tyr 845 phosphorylation.

Phospholipids have recently been discovered to play an important role in cellular regulation. In this study, we focused on phosphatidic acid and lysophosphatidic acid, which are phospholipids known to possess <em>growth</em>-hormonal effects on several types of cells, and examined their <em>growth</em>-promoting effects on murine hair epithelial cells. We discovered that phosphatidic acid possesses intensive <em>growth</em>-promotional effects on hair epithelial cells and epidermal <em>keratinocytes</em>. In contrast, lyso-phosphatidic acid showed lower <em>growth</em>-promoting effects on hair epithelial cells relative to phosphatidic acid and showed minimal or no <em>growth</em>-promoting activity on epidermal <em>keratinocytes</em>. Phosphatidic acid was also shown to have hair-<em>growing</em> activity to induce the anagen phase of the hair cycle in the in vivo murine model. For the purpose of examining the hair-<em>growing</em> mechanisms of phosphatidic acid, we examined its relationship to the mitogen-activated protein kinase cascade linked to cell proliferation and the transforming <em>growth</em> <em>factor</em> beta signal pathway known to be a regulator of catagen induction. We confirmed that phosphatidic acid activates MEK-1/<em>2</em> and upregulates the expression of MEK-1/<em>2</em> in cultured murine hair epithelial cells. Addition of transforming <em>growth</em> <em>factor</em> beta1 to hair epithelial cell cultures concentration-dependently decreased cell <em>growth</em> and induced apoptosis; however, addition of phosphatidic acid to the culture neutralized the <em>growth</em>-inhibiting effects of transforming <em>growth</em> <em>factor</em> beta1 and protected the cells from apoptosis. We speculate that the hair-<em>growing</em> activity of phosphatidic acid is at least linked to its <em>growth</em>-promoting effects on hair epithelial cells that follow mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation and its protective action on transforming-<em>growth</em>-<em>factor</em>-beta1-induced apoptosis that is assumed to trigger catagen induction in the hair cycle.

Exposure of human A431 squamous carcinoma cells to levels of hypoxia found in some solid tumors causes <em>2</em>-fold increases in epidermal <em>growth</em>-<em>factor</em> receptor (EGF-R) mRNA levels and rate of receptor protein synthesis compared with aerobic cells. Similar results are shown for receptor message from other squamous carcinoma cells, human <em>keratinocytes</em>, and human W138 fibroblasts. Less basal tyrosine phosphorylation of the receptor occurs in hypoxic compared with aerobic A431 cells. Scatchard analysis also shows that reoxygenated A431 cells display enhanced surface expression of the EGF-R compared with aerobic control cells. Possible mechanisms and implications for tumor therapy are discussed.

BACKGROUND

Treatment of cancer is increasingly more effective but is associated with short and long term side effects. Oral side effects remain a major source of illness despite the use of a variety of agents to prevent them. One of these side effects is oral mucositis (mouth ulcers).

OBJECTIVE

To evaluate the effectiveness of prophylactic agents for oral mucositis in patients with cancer receiving treatment, compared with other potentially active interventions, placebo or no treatment.

METHODS

Electronic searches of Cochrane Oral Health Group and PaPaS Trials Registers (to 1 June <em>2</em>010), CENTRAL (The Cochrane Library <em>2</em>010, Issue <em>2</em>), MEDLINE via OVID (1950 to 1 June <em>2</em>010), EMBASE via OVID (1980 to 1 June <em>2</em>010), CINAHL via EBSCO (1980 to 1 June <em>2</em>010), CANCERLIT via PubMed (1950 to 1 June <em>2</em>010), OpenSIGLE (1980 to <em>2</em>005) and LILACS via the Virtual Health Library (1980 to 1 June <em>2</em>010) were undertaken. Reference lists from relevant articles were searched and the authors of eligible trials were contacted to identify trials and obtain additional information.

METHODS

Randomised controlled trials of interventions to prevent oral mucositis in patients receiving treatment for cancer.

METHODS

Information regarding methods, participants, interventions, outcome measures, results and risk of bias were independently extracted, in duplicate, by two review authors. Authors were contacted for further details where these were unclear. The Cochrane Collaboration statistical guidelines were followed and risk ratios calculated using random-effects models.

RESULTS

A total of 131 studies with 10,514 randomised participants are now included. Nine interventions, where there was more than one trial in the meta-analysis, showed some statistically significant evidence of a benefit (albeit sometimes weak) for either preventing or reducing the severity of mucositis, compared to either a placebo or no treatment. These nine interventions were: allopurinol, aloe vera, amifostine, cryotherapy, glutamine (intravenous), honey, keratinocyte growth factor, laser, and polymixin/tobramycin/amphotericin (PTA) antibiotic pastille/paste.

CONCLUSIONS

Nine interventions were found to have some benefit with regard to preventing or reducing the severity of mucositis associated with cancer treatment. The strength of the evidence was variable and implications for practice include consideration that benefits may be specific for certain cancer types and treatment. There is a need for further well designed, and conducted trials with sufficient numbers of participants to perform subgroup analyses by type of disease and chemotherapeutic agent.

BACKGROUND

In order to prevent the propagation of genetic mutations, human <em>keratinocytes</em> irradiated with ultraviolet (UV) B light in vitro undergo premature stress-induced senescence or apoptosis. This response to UVB irradiation is dependent on the functional activation of the insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R). Based on this in vitro functional data, we hypothesized that the increased serum levels of insulin in patients with type <em>2</em> diabetes may activate the IGF-1R in skin and lead to a decreased frequency of skin cancer in these patients.

OBJECTIVE

To determine whether the use of insulin by patients with type <em>2</em> diabetes correlated with a change in the incidence in nonmelanoma skin cancer (NMSC).

METHODS

A historical cohort study identifying the incidence of NMSC following the use of two different pharmacological therapies. The patient population was restricted to caucasians who were at least 50 years old when they began the indicated pharmacological therapy. The first group consisted of 1440 patients who used insulin therapy to treat type <em>2</em> diabetes and the second group comprised 4135 patients who used cimetidine to treat their gastrointestinal ailments. An additional group of 6131 patients with diabetes who used noninsulin antidiabetics was added to examine the effect of noninsulin therapies. All patients had regular follow-up visits at the Regenstrief Clinics during the study period between 1980 and 1999. The Regenstrief Clinics is an outpatient facility which serves the general population in Metro-Indianapolis, Indiana, U.S.A.

RESULTS

The incidence of NMSC in patients using insulin was significantly lower than in patients using cimetidine (1.<em>2</em>5% vs. <em>2</em>.35%, P < 0.0<em>2</em>). The decrease in NMSC in patients with type <em>2</em> diabetes correlated specifically with the use of insulin (NMSC incidence insulin-only patients with diabetes: 1.40% vs. those with diabetes using noninsulin therapies: <em>2</em>.35%, P = 0.11).

CONCLUSIONS

Patients using exogenous insulin had a lower risk of developing NMSC and the protective effect of insulin use becomes more distinct with increasing age.

Melanoma <em>growth</em> stimulatory activity/gro alpha (MGSA), a member of the alpha-chemokine family, is produced by a variety of dermal and epidermal cells and can act in a paracrine and autocrine fashion. However, little is known about the importance of MGSA in wound healing. In this study, we quantified the levels of MGSA protein in burn blister and donor site wound fluids. We studied the effects of MGSA on proliferation and migration of primary human <em>keratinocytes</em> and modulation of their integrin expression. Blister fluids contained 0.79 ng/ml (range 0.018 to 4.86 ng/ml) MGSA. Substantial increasing amounts of MGSA were found in donor site fluids from day 1 through day 5 with mean levels ranging from 1.77 to 103 ng/ml at postoperative day 5, which correlated with increasing amounts of tumor necrosis <em>factor</em> alpha (r = 0.86), a known stimulus for MGSA production. In vitro proliferation experiments revealed a maximum stimulation (<em>2</em>.6-fold) with 10 ng/ ml MGSA for 7 days over unstimulated <em>keratinocyte</em> controls; the ED50 was 0.<em>2</em> ng/ml. DNA content analysis revealed an increase in S phase with 10 ng/ml MGSA stimulation. In cultured <em>keratinocytes</em>, MGSA enhanced the mean fluorescence intensity for alpha 6, while no significant change was seen for beta 1, alpha <em>2</em> and alpha 5. We also studied the effect of topically applied MGSA (50 ng/cm<em>2</em>) on the healing of meshed split-thickness human skin grafts on athymic mice. In these wounds, MGSA stimulated the rate of epithelialization (P < 0.05) at day 7, and an increased proportion of mitotic <em>keratinocytes</em> was observed. Wound contraction was significantly (P < 0.05) reduced on days 7 and 14 in the MGSA-treated group. These results suggest that MGSA participates in wound healing by stimulating <em>keratinocyte</em> proliferation.

OBJECTIVE

Progestogen (vaginal progesterone or 17-alpha-hydroxyprogesterone caproate [17OHP-C]) administration to patients at risk for preterm delivery is widely used for the prevention of preterm birth (PTB). The mechanisms by which these agents prevent PTB are poorly understood. Progestogens have immunomodulatory functions; therefore, we investigated the local effects of vaginal progesterone and 17OHP-C on adaptive and innate immune cells implicated in the process of parturition.

METHODS

Pregnant C57BL/6 mice received vaginal progesterone (1 mg per <em>2</em>00 μL, n = 10) or Replens (control, <em>2</em>00 μL, n = 10) from 13 to 17 days postcoitum (dpc) or were subcutaneously injected with 17OHP-C (<em>2</em> mg per 100 μL, n = 10) or castor oil (control, 100 μL, n = 10) on 13, 15, and 17 dpc. Decidual and myometrial leukocytes were isolated prior to term delivery (18.5 dpc) for immunophenotyping by flow cytometry. Cervical tissue samples were collected to determine matrix metalloproteinase (MMP)-9 activity by in situ zymography and visualization of collagen content by Masson's trichrome staining. Plasma concentrations of progesterone, estradiol, and cytokines (interferon [IFN]γ, interleukin (IL)-1β, IL-<em>2</em>, IL-4, IL-5, IL-6, IL-10, IL-1<em>2</em>p70, <em>keratinocyte</em>-activated chemokine/<em>growth</em>-related oncogene, and tumor necrosis <em>factor</em>-α) were quantified by enzyme-linked immunosorbent assays. Pregnant mice pretreated with vaginal progesterone or Replens were injected with 10 μg of an endotoxin on 16.5 dpc (n = 10 each) and monitored via infrared camera until delivery to determine the effect of vaginal progesterone on the rate of PTB.

RESULTS

The following results were found: (1) vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ regulatory T cells; (<em>2</em>) vaginal progesterone, but not 17OHP-C, decreased the proportion of decidual CD8+CD<em>2</em>5+Foxp3+ T cells and macrophages; (3) vaginal progesterone did not result in M1→M<em>2</em> macrophage polarization but reduced the proportion of myometrial IFNγ+ neutrophils and cervical active MMP-9-positive neutrophils and monocytes; (4) 17OHP-C did not reduce the proportion of myometrial IFNγ+ neutrophils; however, it increased the abundance of cervical active MMP-9-positive neutrophils and monocytes; (5) vaginal progesterone immune effects were associated with reduced systemic concentrations of IL-1β but not with alterations in progesterone or estradiol concentrations; and (6) vaginal progesterone pretreatment protected against endotoxin-induced PTB (effect size 50%, P = 0.011).

CONCLUSIONS

Vaginal progesterone, but not 17OHP-C, has local antiinflammatory effects at the maternal-fetal interface and the cervix and protects against endotoxin-induced PTB.

BACKGROUND

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.

METHODS

Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine-glycine-aspartic acid (RGD), 500 μM arginine-glycine-glutamate-serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.

RESULTS

CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.

CONCLUSIONS

Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.

Amphiregulin (AR), an epidermal <em>growth</em> <em>factor</em> (EGF)-like molecule, is a mitogen for <em>keratinocytes</em>. Squamous cell carcinoma (SCC) is a tumor derived from keratinoctyes. Expression of AR mRNA positively correlated with the clinical progression of 39 oral SCC. Oral SCC line, KB, was used as a model to study if increased expression of AR altered the biological behaviors of SCC cells. Exogenous AR dose-dependently enhanced the proliferation of KB cells expressing EGF receptor 1 (EGFR-1), a major receptor for AR, but little AR. Neutralizing anti-AR antibody significantly reversed the stimulatory effect of exogenous AR on KB cell proliferation. Ectopically expressed AR in stable clones manifested higher abilities to proliferate, migrate and invade Matrigel than vector control. Cyclooxygenase <em>2</em> (COX-<em>2</em>), but not metalloprotease 9 (MMP-9) mRNA, was increased in all the AR-expressing stable clones. In summary, AR behaves as a tumor promoter for oral SCC cells partly via increased expression of COX-<em>2</em>.

Acne is a chronic disease hallmarked by sebaceous hyperplasia, follicular hyperkeratosis, and inflammation. Parallel targeting of these <em>factors</em> is required to treat acne effectively. Inhibitors of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) show strong anti-inflammatory effects on immune cells and therapeutic efficacy in autoimmune disorders. Our investigation focused on the expression and functional relevance of these ectopeptidases in three cell types which exhibit an altered phenotype in early acne lesions. We showed for the first time expression of DP IV and APN on human sebocytes. In the SZ95 sebocyte cell line, the DP IV inhibitors Lys[Z(NO<em>2</em>)]-thiazolidide and Lys[Z(NO<em>2</em>)]-pyrrolidide and the APN inhibitors actinonin and bestatin suppressed proliferation, enhanced terminal differentiation, and slightly decreased total neutral lipid production. The anti-inflammatory and differentiation-restoring cytokine IL-1 receptor antagonist was significantly upregulated in SZ95 sebocytes and the HaCaT <em>keratinocyte</em> cell line in the presence of inhibitors. Furthermore, the inhibitors suppressed proliferation and IL-<em>2</em> production of Propionibacterium acnes-stimulated T cells ex vivo and enhanced the expression of the immunosuppressive cytokine transforming <em>growth</em> <em>factor</em>-beta1. Our data provide first evidence for a functional role of DP IV and APN in the sebaceous gland apparatus and for their inhibitors, used alone or in combination, as completely new substances possibly affecting acne pathogenesis in a therapeutic manner.

Ultraviolet radiation (UVR)-induced receptor phosphorylation is increasingly recognized as a widely occurring phenomenon. However, the mechanisms, mediators, and sequence of events involved in this process remain ill-defined. We have recently shown that exposure of human <em>keratinocytes</em> to physiologic doses of ultraviolet B radiation (UVB) activates epidermal <em>growth</em> <em>factor</em> receptor (EGFR)/extracellular-regulated kinase 1 and <em>2</em> (ERK1/<em>2</em>), and p38 signaling pathways via reactive oxygen species. Here we demonstrate that UVB exposure increased intra- and extracellular H<em>2</em>O<em>2</em> production rapidly in a time-dependent manner. An EGFR-specific monoclonal antibody abrogated EGFR autophosphorylation and markedly decreased the phosphorylation of ERK1/<em>2</em> whereas p38 activation was unaffected. Overexpression of catalase strongly inhibited UVB-induced EGFR/ERK1/<em>2</em> pathway activation. These findings establish the sequence of events after UVB irradiation: (i) H<em>2</em>O<em>2</em> generation, (ii) EGFR phosphorylation, and (iii) ERK activation. Our results identify UVB-induced H<em>2</em>O<em>2</em> as a second messenger that is required for EGFR and dependent downstream signaling pathways activation.

BACKGROUND

Oral retinoids have been widely used in psoriasis, but topical forms have been ineffective or irritating.

OBJECTIVE

Our purpose was to determine the clinical and molecular effects of a new topical retinoid, AGN 190168, on psoriasis.

METHODS

Seven patients with psoriasis were treated for <em>2</em> weeks with topical retinoid and <em>2</em> weeks with vehicle. Two control subjects with psoriasis were treated for <em>2</em> weeks with vehicle alone. Biopsy specimens from normal skin as well as from untreated and treated psoriatic lesions were compared by immunohistochemical analysis. Differentiation and inflammatory markers were studied.

RESULTS

Clinical improvement was seen in all seven patients after <em>2</em> weeks of treatment. Improvement was still present, but not significant, after <em>2</em> additional weeks of vehicle application. Histologic examination showed a return to a more normal morphology in four of seven biopsy specimens, which correlated with filaggrin expression. There was a diminution in the precocious expression of keratinocyte transglutaminase, keratin 16, and involucrin, as well as a decrease in epidermal growth factor receptor and in the number of cells expressing intercellular adhesion molecule type 1 and HLA-DR.

CONCLUSIONS

Clinical and histologic improvements were seen in psoriasis in association with the topical application of AGN 190168 at <em>2</em> weeks, including decreased inflammation and restoration of normal epidermal differentiation. Small patient numbers and the possibility that the changes were related to clinical improvement alone and not the topical agent preclude definitive conclusions.

Dickkopf 1 (DKK1), an inhibitor of Wnt signaling, not only functions as a head inducer during development, but also regulates joint remodeling and bone formation, which suggests roles for DKK1 in the pathogenesis of rheumatoid arthritis and multiple myeloma. We recently demonstrated that levels of DKK1 in palmoplantar dermal fibroblasts are physiologically higher than those observed in non-palmoplantar dermal fibroblasts. Thus, the DKK1-rich mesenchyme in palmoplantar dermis affects the overlying epithelium and induces a palmoplantar phenotype in the epidermis. More specifically, DKK1 suppresses melanocyte function and <em>growth</em> through the regulation of microphthalmia-associated transcription <em>factor</em> (MITF) and beta-catenin. Furthermore, DKK1 induces the expression of keratin 9 and alpha-Kelch-like ECT<em>2</em>-interacting protein (alphaKLEIP) but downregulates the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-<em>2</em> (PAR-<em>2</em>) in <em>keratinocytes</em>. Treatment of reconstructed skin with DKK1 reproduces the hypopigmentation and thickening of skin through Wnt/beta-catenin signaling. These studies elucidate why human palmoplantar skin is thicker and paler than non-palmoplantar skin through the secretion of DKK1 by fibroblasts that affect the overlying epidermis. Thus, DKK1 may be useful for reducing skin pigmentation and for thickening photo-aged skin and palmoplantar wounds caused by diabetes mellitus and rheumatic skin diseases.Journal of Investigative Dermatology Symposium Proceedings (<em>2</em>009) 14, 73-75; doi:10.1038/jidsymp.<em>2</em>009.4.

Secondary insults, such as hemorrhagic shock (HS), worsen outcome from traumatic brain injury (TBI). Both TBI and HS modulate levels of inflammatory mediators. We evaluated the addition of HS on the inflammatory response to TBI. Adult male C57BL6J mice were randomized into five groups (n=4 [naïve] or 8/group): naïve; sham; TBI (through mild-to-moderate controlled cortical impact [CCI] at 5 m/sec, 1-mm depth), HS; and CCI+HS. All non-naïve mice underwent identical monitoring and anesthesia. HS and CCI+HS underwent a 35-min period of pressure-controlled hemorrhage (target mean arterial pressure, <em>2</em>5-<em>2</em>7 mm Hg) and a 90-min resuscitation with lactated Ringer's injection and autologous blood transfusion. Mice were sacrificed at <em>2</em> or <em>2</em>4 h after injury. Levels of 13 cytokines, six chemokines, and three <em>growth</em> <em>factors</em> were measured in serum and in five brain tissue regions. Serum levels of several proinflammatory mediators (eotaxin, interferon-inducible protein 10 [IP-10], <em>keratinocyte</em> chemoattractant [KC], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1alpha [MIP-1α], interleukin [IL]-5, IL-6, tumor necrosis <em>factor</em> alpha, and granulocyte colony-stimulating <em>factor</em> [G-CSF]) were increased after CCI alone. Serum levels of fewer proinflammatory mediators (IL-5, IL-6, regulated upon activation, normal T-cell expressed, and secreted, and G-CSF) were increased after CCI+HS. Serum level of anti-inflammatory IL-10 was significantly increased after CCI+HS versus CCI alone. Brain tissue levels of eotaxin, IP-10, KC, MCP-1, MIP-1α, IL-6, and G-CSF were increased after both CCI and CCI+HS. There were no significant differences between levels after CCI alone and CCI+HS in any mediator. Addition of HS to experimental TBI led to a shift toward an anti-inflammatory serum profile--specifically, a marked increase in IL-10 levels. The brain cytokine and chemokine profile after TBI was minimally affected by the addition of HS.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a potent mitogen of pulmonary bronchial and alveolar epithelial cells. However, it is unclear which type(s) of airway epithelial cells (AEC) proliferate(s) in response to KGF. AEC proliferation was induced in rats by either endobronchial instillation of 5 mg recombinant human (rHu) KGF per kg body weight or by adenoviral transfer of the human KGF gene (Ad5-HuKGF). Alterations in terminal airway AEC were followed for up to 7 days after rHuKGF, and for up to <em>2</em>8 days after Ad5-HuKGF. Cell proliferation, as assessed by immunohistochemistry (IHC) for incorporated 5-bromo-<em>2</em>'-deoxyuridine (BrdU) and quantified by stereology, peaked at days 1-<em>2</em> and was resolved by day 7 after rHuKGF and by day <em>2</em>1 after Ad5-HuKGF. Double immunofluorescence labelling for BrdU or Ki-67 on the one hand, and for Clara cell specific protein 10 (CC10) and calcitonin-gene related peptide on the other hand, demonstrated that Clara cells, not pulmonary neuroendocrine cells, proliferated in response to human KGF. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) method in conjunction with IHC for MNF116 failed to detect significant numbers of apoptotic AEC. IHC in conjunction with stereology revealed transient phenotypic alterations with a decrease in CC10, an increase in surfactant protein D and an increase in CD44v6 in AEC. The authors conclude that Clara cells responded to human <em>keratinocyte</em> <em>growth</em> <em>factor</em> in vivo by proliferation as well as by changes in protein expression, whereas no significant response was observed in pulmonary neuroendocrine cells. As Clara cells are intimately involved in airway epithelial repair, ion and fluid transport, and modulate lung inflammation, the potential of human <em>keratinocyte</em> <em>growth</em> <em>factor</em> to protect the lung may in part rely on the response of Clara cells.

Oxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in many human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT <em>keratinocyte</em> cell culture model, we investigated redox regulation and inflammation to periodic, low-dose oxidative stress after two, six, eight, ten, and twelve weeks. Chronic redox stress was generated by recurrent incubation with cold physical plasma-treated cell culture medium. Using transcriptome microarray technology, we identified both acute ROS-stress responses as well as numerous adaptions after several weeks of redox challenge. We determined a differential expression (<em>2</em>-fold, FDR < 0.01, p < 0.05) of <em>2</em>60 genes that function in inflammation and redox homeostasis, such as cytokines (e.g., IL-6, IL-8, and IL-10), <em>growth</em> <em>factors</em> (e.g., CSF<em>2</em>, FGF, and IGF-<em>2</em>), and antioxidant enzymes (e.g., HMOX, NQO1, GPX, and PRDX). Apoptotic signaling was affected rather modestly, especially in p53 downstream targets (e.g., BCL<em>2</em>, BBC3, and GADD45). Strikingly, the cell-protective heat shock protein HSP<em>2</em>7 was strongly upregulated (p < 0.001). These results suggested cellular adaptions to frequent redox stress and may help to better understand the inflammatory responses in redox-related diseases.

Transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1) is an important modulator of skin morphogenesis and cutaneous wound repair. To gain insight into the mechanisms of TGF-beta1 action in the skin, we used the differential display RT-PCR technique to identify genes that are regulated by this <em>factor</em> in cultured human <em>keratinocytes</em>. We obtained several partial cDNA clones. One of them was identical to the 3'-end of p11, the small and regulatory subunit of the calpactin I complex [(annexin II)<em>2</em>(p11)<em>2</em>]. RNase protection and northern blot analysis revealed specific regulation of expression of both subunits of this heterotetrameric protein (p11 and annexin II) by TGF-beta1 as well as by other <em>growth</em> <em>factors</em>, although the time course and degree of induction or suppression were different for each gene. Furthermore, we analyzed p11 and annexin II expression in normal and wounded skin. Both p11 and annexin II mRNAs were found in the dermal and epidermal compartments of normal human skin. Immunohistochemical studies demonstrated the presence of p11 at equally high levels in all layers of normal epidermis and in the hyper-proliferative epithelium at the wound edge. By contrast, annexin II expression was high in the basal layer of normal epidermis but low in the suprabasal layers and in the hyper-proliferative epithelium at the wound edge, suggesting a differentiation-specific regulation of this calpactin I subunit. The differential expression and regulation of p11 and annexin II subunits in <em>keratinocytes</em> suggest the existence of different ratios of monomeric versus p11-complexed annexin II that might be associated with different cellular functions.

Fumaric acid esters (FAEs) such as dimethylfumarate (DMF) are used for the treatment of adults with moderate-to-severe psoriasis. The mode of action of FAEs is complex. Here, we provide a comprehensive review of the literature to describe the molecular mechanisms by which DMF and its active metabolite monomethylfumarate (MMF) exert their anti-inflammatory and immune modulatory effects. MMF can bind to the hydroxy-carboxylic acid receptor <em>2</em> (HCA<em>2</em>) on the cell surface and both DMF and MMF react with intracellular glutathione following cell penetration. DMF and to some extent also MMF modulate the activity of certain cellular signalling proteins such as the nuclear <em>factor</em> (erythroid-derived <em>2</em>)-like <em>2</em> (Nrf<em>2</em>), nuclear <em>factor</em> kappa B (Nf-κB) and the cellular concentration of cyclic adenosine monophosphate. Some studies show that DMF can also affect the hypoxia-inducible <em>factor</em> 1-alpha (HIF-1α). These actions seem to be responsible for i) the downregulation of inflammatory cytokines and ii) an overall shift from a proinflammatory Th1/Th17 response to an anti-inflammatory/regulatory Th<em>2</em> response. Both steps are necessary for the amelioration of psoriatic inflammation, although additional mechanisms have been proposed. There is a <em>growing</em> body of evidence to support the notion that DMF/MMF may also exert effects on granulocytes and non-immune cell lineages including <em>keratinocytes</em> and endothelial cells. A better understanding of the multiple molecular mechanisms involved in the cellular action of FAEs will help to adapt and further improve the use of such small molecules for the treatment of psoriasis and other chronic inflammatory diseases.