The airway epithelium may be damaged by inhalation of noxious agents, in response to pathogens, or during endotracheal intubation and mechanical ventilation. Maintenance of an intact epithelium is important for lung fluid balance, and the loss of epithelium may stimulate inflammatory responses. Epithelial repair in the airways following injury must occur on a substrate that undergoes cyclic elongation and compression during respiration. We have previously shown that cyclic mechanical strain inhibits wound closure in the airway epithelium (Savla and Waters, 1998b). In this study, we investigated the stimulation of epithelial wound closure by <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) in vitro and the mechanisms by which KGF overcomes the inhibition due to mechanical strain. Primary cultures of normal human bronchial epithelial cells (NHBE) and a cell line of human airway epithelial cells, Calu 3, were grown on Silastic membranes, and a wound was scraped across the well. The wells were then exposed to cyclic strain using the Flexercell Strain Unit, and wound closure was measured. While cyclic elongation (<em>2</em>0% maximum) and cyclic compression (approximately <em>2</em>%) both inhibited wound closure in untreated wells, treatment with KGF (50 ng/ml) significantly accelerated wound closure and overcame the inhibition due to cyclic strain. Since wound closure involves cell spreading, migration, and proliferation, we investigated the effect of cyclic strain on cell area, cell-cell distance, and cell velocity at the wound edge. While the cell area increased in unstretched monolayers, the cell area of monolayers in compressed regions decreased significantly. Treatment with KGF increased the cell area in both cyclically elongated and compressed cells. Also, when cells were treated with KGF, cell velocity was significantly increased in both static and cyclically strained monolayers, and cyclic strain did not inhibit cell migration. These results suggest that KGF is an important <em>factor</em> in epithelial repair that is capable of overcoming the inhibition of repair due to physiological levels of cyclic strain.

BACKGROUND

Massive small bowel resection with subsequent short bowel syndrome (SBS) leads to the acute loss of epithelial cell (EC) absorptive function. Keratinocyte growth factor (KGF) has been shown to improve EC growth, although little is known about KGF activity on EC function after SBS. We hypothesized that KGF would improve epithelial function in a mouse SBS model.

METHODS

Adult C57BL/6J mice were randomized to a 55% mid-small bowel resection (SBS), SBS with KGF administration (SBSKGF), or sham-operated (Control) group, and were killed at 7 days. Ussing chambers were used to study epithelial function. Short circuit current (Isc) was monitored. EC absorption was studied by measuring (1) glucose [3-O-methyl-D-[1-3H]glucose (3-OMG)] absorption; (2) sodium coupled amino acid (alanine) absorption; and (3) changes in Isc by using the absorptive agent D-glucose (stimulated Na+ absorption). Epithelial barrier function was measured with transepithelial resistance (TER) and transmural passage of 3H-mannitol (Papp). ECs were separated along the crypt-villus axis with laser capture microdissection. Epithelial KGF receptor (KGFR) mRNA expression was studied using real time reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

KGF administration increased the basic ion transport activity and net transepithelial absorption of 3-OMG and sodium-coupled alanine absorption. SBS significantly decreased epithelial ion transport, including the Na+ absorption stimulated by D-glucose and L-alanine. KGF administration partially improves Na+ absorption. KGF had no apparent effect on the TER and 3H-mannitol permeability in this study. KGF upregulated EC KGFR mRNA expression, predominately in the crypt and lower portion of the villus.

CONCLUSIONS

KGF administration improves epithelial absorptive function and stimulates intestinal proliferation after SBS. This suggests that KGF improves intestinal adaptation after SBS and may have clinical applicability.

In the mouse <em>keratinocyte</em> line HEL-30 the epidermal mitogen transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) stimulated the rapid release of arachidonic acid in a dose- and time-dependent manner. The liberation of arachidonic acid was due to the activation of a Ca(<em>2</em>+)-dependent cytosolic phospholipase A<em>2</em> (cPLA<em>2</em>). The activation mechanism critically depended on a functionally active epidermal <em>growth</em> <em>factor</em> receptor tyrosine kinase and occurred independently of phospholipase C-mediated increases in cellular diacylglycerol and inositol 1,4,5-trisphosphate concentrations and protein kinase C activation. The activation included an increase in cytosolic PLA<em>2</em> (cPLA<em>2</em>) activity and an association of the enzyme with the membrane fraction. Both activation steps apparently occurred in the presence of basal cytoplasmic Ca<em>2</em>+ concentrations. Moreover, cPLA<em>2</em> or a closely associated protein was found to be phosphorylated on tyrosine upon TGF-alpha challenge of the cells. The data suggest that tyrosine phosphorylation is involved in the TGF-alpha-induced activation of cPLA<em>2</em>.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell <em>growth</em> and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(<em>2</em>) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into <em>keratinocytes</em> markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (4<em>2</em>% closed versus 9<em>2</em>% in controls, P < 0.05) and homozygous (<em>2</em>5% versus 9<em>2</em>%, P < 0.01) animals at day 1<em>2</em> after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> isoform b (FGFR<em>2</em>-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR<em>2</em>-IIIb in hepatocellular carcinoma (HCC). FGFR<em>2</em>-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR<em>2</em>-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR<em>2</em>-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-<em>2</em>IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR<em>2</em> expression in a subset of HCC cells. FGFR<em>2</em>-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR<em>2</em>-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR<em>2</em>-IIIb expression in HCC cells were not affected by <em>keratinocyte</em> <em>growth</em> <em>factor</em> or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR<em>2</em>-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.

Autocrine epidermal <em>growth</em> <em>factor</em> receptor activation by transforming <em>growth</em> <em>factor</em> alpha (TGF alpha) has been implicated in <em>growth</em> stimulation during epithelial neoplasia. Using <em>keratinocytes</em> isolated from mice with genetic defects in TGF alpha expression, we tested whether TGF alpha is required for transformation by the v-rasHa oncogene. Introduction of v-rasHa into primary epidermal cultures using a retroviral vector stimulated <em>growth</em> of both control (TGF alpha +/+, BALB/c) and TGF alpha-deficient (TGF alpha -/-, wa-1) <em>keratinocytes</em>. Moreover, v-rasHa elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal <em>growth</em> <em>factor</em> (EGF) receptor activation, in both TGF alpha +/+ and TGF alpha -/- <em>keratinocytes</em>. v-rasHa markedly increased secreted >> 10-fold) and cell-associated (<em>2</em>-3-fold) TGF alpha levels in <em>keratinocytes</em> from TGF alpha +/+ and BALB/c mice, but not TGF alpha -/- or wa-1 mice. Based on Northern blot analysis, v-rasHa induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like <em>growth</em> <em>factor</em>, and betacellulin in cultured <em>keratinocytes</em> from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGF alpha transcript, wa-1 <em>keratinocytes</em> expressed two additional TGF alpha transcripts, 4.7 and 5.<em>2</em> kilobases long. All three transcripts were up-regulated in response to v-rasHa, as well as exogenous TGF alpha or <em>keratinocyte</em> <em>growth</em> <em>factor</em> treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-rasHa <em>keratinocytes</em> from control as well as TGF alpha-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like <em>growth</em> <em>factor</em>, and betacellulin mRNA, regardless of their TGF alpha status. These findings indicate that TGF alpha is not essential for epidermal neoplasia induced by the v-rasHa oncogene and suggest that another EGF family member(s) may contribute to autocrine <em>growth</em> stimulation of ras-transformed <em>keratinocytes</em>.

OBJECTIVE

Keratinocyte growth factor (KGF) has potent mitogenic activity on normal epithelial cells and has been found to enhance intestinal crypt cell survival in irradiated mice and to prevent radiation and chemotherapy-induced mucositis in animal models. The purpose of the study reported here is to investigate the effect of recombinant human KGF on the proliferation and survival of human squamous carcinoma cell lines following irradiation.

METHODS

The level of KGF receptor (KGFR) mRNA in normal Balb/Mk cell line and human head and neck squamous carcinoma cell lines was assessed using a RNase protection assay. The clonogenic assay and MTT assay were used to study the proliferative effects of KGF on human tumor cell lines and Balb/MK cell line in vitro. Effects of KGF on in vivo tumor growth and radiosensitivity were studied in three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice.

RESULTS

Seven of 10 tumor cell lines studied expressed KGFR mRNA. None of these tumor cell lines showed enhanced proliferation when exposed to KGF for 2 days or less. Prolonged exposure to KGF for 7 days or longer resulted in low level stimulation of proliferation in both clonogenic and MTT assays in four of seven KGFR-positive cell lines. Two KGFR-negative cell lines also had a low proliferative response to KGF in a clonogenic assay, but not in the MTT assay. Normal keratinocyte Balb/MK cells, which expressed a moderate level of KGFR mRNA, had a strongly proliferative response to KGF. Its KGF enhancement ratio (KER) of plating efficiency was 24-70 times higher than that of the tumor cells studied (p < 0.001). The KGF-stimulated tumor cell growth was almost completely inhibited by heparin or epidermal growth factor (EGF). There were no significant differences (p>> 0.05) in the survival of any of tumor cell lines in the presence or absence of KGF (100 ng/ml) irradiated with doses of 0-15 Gy, and no significant differences (p>> 0.05) between the radiobiological parameters D0, Dq, and n number from the SHMT model, alpha, beta, and alpha/beta ratio from the LQ model and SF2 for radiation survival curves for cell lines irradiated in the presence or absence of KGF. Three KGFR-positive human squamous cell carcinoma xenografts in nude mice, and a murine KGFR-negative melanoma tumor in Balb/c mice treated with 1.0 mg/kg of KGF for 3 days grew at the same rate as in untreated mice.

CONCLUSIONS

The recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of cancers.

During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of <em>keratinocytes</em> in response to transforming <em>growth</em> <em>factor</em> β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/<em>2</em> (Erk1/<em>2</em>) activation. TGFβ-treated sheets display a spatial pattern of Erk1/<em>2</em> activation in which the highest levels of Erk1/<em>2</em> activity are concentrated toward the leading edge of a sheet. We show that Erk1/<em>2</em> activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/<em>2</em> activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/<em>2</em> activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/<em>2</em> throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/<em>2</em> activity) can influence the guidance of a collective group of cells during wound healing.

Human <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a recently identified mitogen for epithelial cells produced by normal stromal fibroblasts. KGF has been shown to stimulate <em>keratinocyte</em> migration and promote re-epithelialization of skin suggesting a critical role for KGF in wound healing. To understand how KGF might be regulated during wound healing, we examined the ability of the pro-inflammatory cytokines interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) interleukin-6 (IL-6), tumor necrosis <em>factor</em>-alpha (TNF-alpha) transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) and interferon-gamma (IFN-gamma) to modulate KGF gene expression in cultured human fibroblasts, using northern blot analysis. Exposure to IL-1 alpha (<em>2</em>0 units/ml) or IL-1 beta (100 units/ml) for <em>2</em>4 h increased KGF mRNA expression by 35<em>2</em>% and 504%, respectively, with early induction seen at <em>2</em> h and maximal induction seen at 8 h. TNF-alpha (30 ng/ml) increased KGF mRNA expression by 535% at <em>2</em>4 h, with induction first seen at 8 h. The maximal induction of KGF mRNA was observed when IL-1 alpha, IL-1 beta and TNF-alpha were used at 100 units/ml, and 3 ng/ml, respectively, although concentrations 100-500-fold lower (IL-1 alpha, 0.0<em>2</em> units/ml; IL-beta, 0.0<em>2</em> units/ml; and TNF-alpha, 0.03 ng/ml) were nearly as stimulatory, increasing KGF mRNA expression by 175%, <em>2</em>54% and 3<em>2</em><em>2</em>%, respectively. IL-6 (<em>2</em>00 units/ml), TGF-beta 1 (5 ng/ml) and IFN-gamma (<em>2</em>00 units/ml) did not change the level of KGF mRNA at <em>2</em>4 h in human fibroblasts under the same conditions. The protein synthesis inhibitor cycloheximide abrogated the effects of IL-1 alpha, IL-1 beta and TNF-alpha on KGF gene induction, indicating that new protein synthesis is required in the process. Dexamethasone (10(-7) M), known to inhibit inflammatory reactions and retard wound healing, also inhibited the induction of KGF mRNA expression by IL-1 alpha, IL-1 beta and TNF-alpha. Individual variation in KGF mRNA expression was see when fibroblasts from different aged donors were analysed, but no consistent age-associated change was observed. These results suggest that IL-1 alpha, IL-1 beta and TNF-alpha up-regulate KGF gene expression in fibroblasts and might be responsible for its induction following skin wounding or other injury.

Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-<em>2</em> in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial <em>growth</em> <em>factor</em> in <em>keratinocytes</em> and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal <em>keratinocytes</em> (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase-PCR, and western blotting revealed marked overexpression of complement <em>factor</em> I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell-specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal-regulated kinase (ERK) 1/<em>2</em> activation. Knockdown of CFI expression potently inhibited <em>growth</em> of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer.

BACKGROUND

A feature of psoriasis is the rapid proliferation of <em>keratinocytes</em>, during which apoptosis is blocked and angiogenesis starts. It is known that tumor hypoxic cells produce histone deacetylase-1 (HDAC-1), which up-regulates hypoxia-inducible <em>factor</em>-1alpha (HIF-1alpha) and down-regulates von Hippel-Lindau (VHL) protein by up-regulating vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression. It has been reported recently that the porcine peptide PR39 (homologous to human LL-37) has angiogenic and antiapoptotic activity. Thus, LL-37, induced by insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), could help in the production of VEGF. PR39 also induces the expression of inhibitor of apoptosis protein-<em>2</em> (IAP-<em>2</em>), which blocks apoptosis. The purpose of this work was to analyze whether these genes and their proteins are expressed in psoriatic biopsies.

METHODS

Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) messenger RNA (mRNA) expression and immunohistochemical staining, we studied VHL, IAP-<em>2</em>, and related genes in skin biopsies from psoriatic patients and healthy subjects.

RESULTS

An over-expression of the mRNA for HDAC-1, HIF-1alpha, LL-37, and IGF-1 in psoriatic skin, in comparison with skin from healthy subjects, was found. The antiangiogenic VHL mRNA and protein were under-expressed in psoriatic skin and highly expressed in healthy skin. The antiapoptotic IAP-<em>2</em> was over-expressed in dermal endothelial cells from psoriatic skin. The pro-apoptotic Bax, Fas, and FasL mRNAs were expressed.

CONCLUSIONS

These findings suggest that there could be an association of HDAC-1, HIF-1alpha, LL-37, VHL, and IAP-<em>2</em> with angiogenic and apoptotic mechanisms in psoriasis.

Nerve <em>growth</em> <em>factor</em> (NGF) is produced by <em>keratinocytes</em> and modulates their proliferation and apoptosis. However, it is as yet unknown whether other members of the NGF family of neurotrophins, brain-derived neurotrophic <em>factor</em> (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), also modulate <em>keratinocyte</em> proliferation in situ. We determined by ELISA and reverse transcriptase-PCR that BDNF, NT-3, and NT-4 are expressed in C57BL/6 mouse skin. By immunofluorescence, the subcutaneous panniculus carnosus muscle and arrector pili muscle showed strong NT-3 immunoreactivity, whereas BDNF-IR was found only in skin nerve bundles. NT-4 immunoreactivity was noted in single epidermal <em>keratinocytes</em>. The high affinity receptor for both BDNF and NT-4, TrkB, was detected in basal and suprabasal epidermal <em>keratinocytes</em>, whereas the high affinity NT-3 receptor, TrkC, was observed in skin nerve bundles. Compared with the corresponding age-matched wild-type mice, BDNF or NT-3-overexpressing transgenic mice showed a significantly increased epidermal thickness and enhanced number of Ki-67-positive (ie, proliferating) epidermal <em>keratinocytes</em> in vivo, whereas the number of these cells was substantially reduced in BDNF knockout mice. In skin organ culture of C57BL/6 mice, BDNF, NT-3, and NT-4 all significantly increased 5-bromo-<em>2</em>'-deoxyuridine incorporation into epidermal <em>keratinocytes</em>. Co-administration of NGF neutralizing antibody failed to abrogate the stimulatory effect of NT-3 on <em>keratinocyte</em> proliferation in skin organ culture. This demonstrates that normal murine epidermal <em>keratinocytes</em> in situ are direct or indirect target cells for these neurotrophins. Therefore, BDNF, NT-3, and NT-4 can also act as "epitheliotrophins" and may thus be intimately involved in the control of epidermal homeostasis.

To help elucidate the <em>factors</em> responsible for the infundibular changes seen in acne, the human sebaceous pilosebaceous infundibulum was isolated by microdissection and maintained for 7 d in <em>keratinocyte</em> serum-free medium supplemented with 50 micrograms/ml bovine pituitary extract, 100 units/ml penicillin and streptomycin, <em>2</em>.5 micrograms/ml amphotericin B and CaCl<em>2</em>(10H<em>2</em>O) to give a final Ca<em>2</em>+ concentration of <em>2</em> mM. Infundibular structure was maintained over 7 d in this medium; the pattern of cell division mimicked that in vivo. The rate of cell division was significantly higher than previously described for infundibula maintained in supplemented William's E medium, and moreover did not fall over 7 d. The addition of 1 ng/ml interleukin-1 alpha (IL-1 alpha) caused hypercornification of the infundibulum similar to that seen in comedones; this could be blocked by 1000 ng/ml interleukin-1 receptor antagonist (IL-1ra). In about <em>2</em>0% of subjects there was spontaneous hypercornification of the infundibulum that could be blocked by 1000 ng/ml IL-1ra, suggesting that the infundibulum is capable of synthesising IL-1 alpha. The addition of 5 ng/ml epidermal <em>growth</em> <em>factor</em> or 5 ng/ml transforming <em>growth</em> <em>factor</em>-alpha to the medium caused a disorganisation of the <em>keratinocytes</em> of the infundibulum that resulted in rupturing similar to that seen in the more severe, purulent grades of acne. The addition of 1 microM 13-cis retinoic acid caused a significant reduction in the rate of DNA synthesis and apparent parakeratosis. We are now, therefore, able to model histologically the major infundibular changes in acne.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>, repifermin) is a homolog of KGF-1 with epithelial mitogenic activities. We investigated the therapeutic role of KGF-<em>2</em> in intestinal ulceration and its mechanisms of protection. KGF-<em>2</em> (0.3-5 mg/kg) was administered before or after induction of small intestinal ulceration by indomethacin (Indo) in prevention and treatment protocols. In acute studies, KGF-<em>2</em> was injected for up to 7 days before or daily for 5 days after Indo. In a 15-day chronic study, KGF-<em>2</em> was injected intravenously daily beginning before or 7 days after Indo. Injury was evaluated by blinded macroscopic and microscopic inflammatory scores, epithelial BrdU staining, tissue IL-1beta, PGE(<em>2</em>), and hydroxyproline concentrations, and collagen type I RNA expression. In vitro effects of KGF-<em>2</em> were evaluated by epithelial cellular proliferation, restitution of wounded monolayers, PGE(<em>2</em>) secretion, and expression of COX-<em>2</em> and collagen mRNA. Intravenous KGF-<em>2</em> significantly decreased acute intestinal injury by all parameters and significantly decreased chronic ulceration. Pretreatment, daily infusion, and delayed treatment were effective. KGF-<em>2</em> promoted in vitro epithelial restitution with only modest effects on epithelial cell proliferation, stimulated COX-<em>2</em> expression in cultured epithelial cells, and upregulated in vitro and in vivo PGE(<em>2</em>) production. KGF-<em>2</em> did not affect in vivo fibrosis, although it induced collagen expression in cultured intestinal myofibroblasts. These results suggest that KGF-<em>2</em> inhibits intestinal inflammation by stimulating epithelial restitution and protective PGs.

The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal <em>keratinocytes</em> as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified <em>keratinocyte</em> cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (<em>growth</em> <em>factors</em>, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human <em>keratinocytes</em>: mechanical injuries (i) immediately stimulate the tyrosine phosphorylation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p<em>2</em>1ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/<em>2</em>, c-Jun NH<em>2</em> terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription <em>factor</em> 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription <em>factor</em> complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human <em>keratinocyte</em> cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from <em>2</em>7 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 1<em>2</em> sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into <em>2</em> distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX<em>2</em>, MSX<em>2</em>, DLX<em>2</em>, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive <em>growth</em>, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX<em>2</em> was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription <em>factors</em>. In contrast, gene expression of KCOT indicates differentiation toward <em>keratinocytes</em>.

Acute lung injury (ALI) affects over 10% of patients hospitalised in critical care, with acute respiratory distress syndrome (ARDS) being the most severe form of ALI and having a mortality rate in the region of 40%. There has been slow but incremental progress in identification of biomarkers that contribute to the pathophysiology of ARDS, have utility in diagnosis and monitoring, and that are potential therapeutic targets (Calfee CS, Delucchi K, Parsons PE, Thompson BT, Ware LB, Matthay MA, Thompson T, Ware LB, Matthay MA, Lancet Respir Med <em>2</em>014, <em>2</em>:611--6<em>2</em>0). However, a major issue is that ARDS is such a heterogeneous, multi-<em>factor</em>ial, end-stage condition that the strategies for "lumping and splitting" are critical (Prescott HC, Calfee CS, Thompson BT, Angus DC, Liu VX, Am J Respir Crit Care Med <em>2</em>016, 194:147--155). Nevertheless, sequencing of the human genome, the availability of improved methods for analysis of transcription to mRNA (gene expression), and development of sensitive immunoassays has allowed the application of network biology to ARDS, with these biomarkers offering potential for personalised or precision medicine (Sweeney TE, Khatri P, Toward precision medicine Crit Care Med; <em>2</em>017 45:934-939). Biomarker panels have potential applications in molecular phenotyping for identifying patients at risk of developing ARDS, diagnosis of ARDS, risk stratification and monitoring. Two subphenotypes of ARDS have been identified on the basis of blood biomarkers: hypo-inflammatory and hyper-inflammatory. The hyper-inflammatory subphenotype is associated with shock, metabolic acidosis and worst clinical outcomes. Biomarkers of particular interest have included interleukins (IL-6 and IL-8), interferon gamma (IFN-γ), surfactant proteins (SPD and SPB), von Willebrand <em>factor</em> antigen, angiopoietin 1/<em>2</em> and plasminogen activator inhibitor-1 (PAI-1). In terms of gene expression (mRNA) in blood there have been found to be increases in neutrophil-related genes in sepsis-induced and influenza-induced ARDS, but whole blood expression does not give a robust diagnostic test for ARDS. Despite improvements in management of ARDS on the critical care unit, this complex disease continues to be a major life-threatening event. Clinical trials of β_<em>2</em>-agonists, statins, surfactants and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) have been disappointing. In addition, monoclonal antibodies (anti-TNF) and TNFR fusion protein have also been unconvincing. However, there have been major advances in methods of mechanical ventilation, a neuromuscular blocker (cisatracurium besilate) has shown some benefit, and stem cell therapy is being developed. In the future, by understanding the role of biomarkers in the pathophysiology of ARDS and lung injury, it is hoped that this will provide rational therapeutic targets and ultimately improve clinical care (Seymour CW, Gomez H, Chang CH, Clermont G, Kellum JA, Kennedy J, Yende S, Angus DC, Crit Care <em>2</em>017, <em>2</em>1:<em>2</em>57).

The functions of transforming <em>growth</em> <em>factor</em> beta-1(TGFbeta1) are cell-context specific. We have found that TGFbeta1 expression in human skin squamous cell carcinoma (SCC) samples has two distinct distribution patterns: (1) either predominantly in suprabasal layers or (<em>2</em>) throughout tumor epithelia including basal proliferative cells. To understand whether the spatial TGFbeta1 expression patterns affect its functions, we have generated several <em>keratinocyte</em>-specific transgenic mouse models in which TGFbeta1 overexpression can be induced either predominantly in the suprabasal epidermis or in the basal layer of the epidermis and hair follicles. Suprabasal TGFbeta1 overexpression inhibits <em>keratinocyte</em> proliferation, suppresses skin carcinogenesis at early stages, but promotes tumor invasion at later stages. In contrast, TGFbeta1 overexpression in the basal layer of the epidermis and hair follicles causes a severe inflammatory skin disorder and epidermal hyperproliferation. Given the importance of inflammation in cancer development, our data suggest that TGFbeta1-induced skin inflammation may override its tumor suppressive effect at early stages during skin carcinogenesis. This hypothesis is further suggested by our recent study that Smad3 knockout mice are resistant to skin chemical carcinogenesis at least in part via abrogation of endogenous TGFbeta1-induced inflammation. This review intends to summarize current insights into the role of TGFbeta1 in skin inflammation and carcinogenesis.

There are about 600 million betel quid (BQ) chewers in the world. BQ chewing is associated with increased incidence of oral cancer and submucous fibrosis. In this study, areca nut (AN) extract (<em>2</em>00-800 microg/ml) induced the prostaglandin E(<em>2</em>) (PGE(<em>2</em>)) production by 1. 4-3.4-fold and 6-keto-PGF(1 alpha) production by 1.1-1.7-fold of gingival <em>keratinocytes</em> (GK), respectively, following <em>2</em>4 h of exposure. Exposure of GK to AN extract (>400 microg/ml) led to cell retraction and intracellular vacuoles formation. At concentrations of 800 and 1<em>2</em>00 microg/ml, AN extract induced cell death at <em>2</em>1-<em>2</em>4 and 3<em>2</em>-5<em>2</em>% as detected by MTT assay and cellular lactate dehydrogenase release, respectively. Interestingly, AN-induced morphological changes of GK are reversible. GK can still proliferate following exposure to AN extract. Cytotoxicity of AN extract cannot be inhibited by indomethacin (1 microM) and aspirin (50 microM), indicating that prostaglandin (PG) production is not the major <em>factor</em> responsible for AN cytotoxicity. PGE(<em>2</em>) exhibited little effect on the <em>growth</em> of GK at concentrations ranging from 100-1000 pg/ml. Stimulating GK production of PGs by AN extract could be due to induction of cyclooxygenase-<em>2</em> (COX-<em>2</em>) mRNA expression and protein production. These results suggest that AN ingredients are critical in the pathogenesis of oral submucous fibrosis and oral cancer via their stimulatory effects on the PGs, COX-<em>2</em> production and associated tissue inflammatory responses. AN cytotoxicity to GK is not directly mediated by COX-<em>2</em> stimulation and PG production.

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human <em>keratinocytes</em> were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial <em>growth</em> <em>factor</em>, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial <em>growth</em> <em>factor</em>-modified or control <em>keratinocytes</em> were prepared, and were cultured in vitro for <em>2</em>1 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial <em>growth</em> <em>factor</em> mRNA in genetically modified <em>keratinocytes</em> and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial <em>growth</em> <em>factor</em>-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial <em>growth</em> <em>factor</em> protein throughout the entire culture period. The bioactivity of vascular endothelial <em>growth</em> <em>factor</em> protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell <em>growth</em> assay. Vascular endothelial <em>growth</em> <em>factor</em>-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial <em>growth</em> <em>factor</em> mRNA expression was detected in the modified grafts for at least <em>2</em> wk after surgery. Vascular endothelial <em>growth</em> <em>factor</em>-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of <em>keratinocytes</em> in cultured skin substitutes can lead to increased vascular endothelial <em>growth</em> <em>factor</em> expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.

Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) overexpression is an unfavorable prognostic marker in laryngeal squamous cell carcinoma (SCC). EGFR stimulates cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression in normal human <em>keratinocytes</em> and squamous carcinoma cells. Based on these observations a prognostic role of COX-<em>2</em> expression in laryngeal SCC can be hypothesized. Consequently, COX-<em>2</em> expression was studied in laryngeal SCC (median follow-up = 47 months; range: <em>2</em>-87 months) by quantitative immunohistochemistry (n = 61) and EGFR by binding assay (n = 51). Well-differentiated regions of laryngeal SCC revealed strong COX-<em>2</em> immunostaining, whereas histologically normal areas neighboring tumor as well as poorly-differentiated tumors were negative. Immunohistochemical results were confirmed by Western blot analyses. Cox's regression analysis showed that the combination of low levels of COX-<em>2</em> integrated density and high levels of EGFR covariates provided strong prediction, at 5-year follow-up, of both poor overall survival (chi(<em>2</em>) = 1<em>2</em>.905; p = 0.0016) and relapse-free survival (chi(<em>2</em>) = 9.<em>2</em>09; p = 0.01). In vitro studies on CO-K3 cell line, obtained from an EGFR positive, COX-<em>2</em> negative poorly-differentiated laryngeal SCC, revealed that EGF stimulation failed to induce COX-<em>2</em> expression and PGE<em>2</em> production suggesting a change in EGFR signaling pathway. These findings indicate that COX-<em>2</em> is overexpressed in less aggressive, low grade laryngeal SCC, whereas its expression is lost when tumors progress to a more malignant phenotype.

We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse <em>keratinocyte</em> and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced <em>keratinocyte</em> and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic <em>growth</em> responses in skin epidermis through secretion of heparin-binding EGF-like <em>growth</em> <em>factor</em>, GM-CSF, IL-1α, and cyclooxygenase-<em>2</em> and activation of mitogen-activated protein kinase pathways. We identified altered expression of several <em>keratinocyte</em>-derived mitogenic paracrine <em>growth</em> <em>factors</em> such as endothelin 1, hepatocyte <em>growth</em> <em>factor</em>, α-melanocyte stimulating hormone, stem cell <em>factor</em>, and fibroblast <em>growth</em> <em>factor</em>-<em>2</em> in skin of mice lacking RXRα in epidermal <em>keratinocytes</em> (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

Mast cells (MCs), apart from their classic role in allergy, contribute to a number of biologic processes including wound healing. In particular, two aspects of their histologic distribution within the skin have attracted the attention of researchers to study their wound healing role; they represent up to 8% of the total number of cells within the dermis and their cutaneous versions are localized adjacent to the epidermis and the subdermal vasculature and nerves. At the onset of a cutaneous injury, the accumulation of MCs and release of proinflammatory and immunomodulatory mediators have been well documented. The role of MC-derived mediators has been investigated through the stages of wound healing including inflammation, proliferation, and remodeling. They contribute to hemostasis and clot formation by enhancing the expression of <em>factor</em> XIIIa in dermal dendrocytes through release of TNF-α, and contribute to clot stabilization. <em>Keratinocytes</em>, by secreting stem cell <em>factor</em> (SCF), recruit MCs to the site. MCs in return release inflammatory mediators, including predominantly histamine, VEGF, interleukin (IL)-6, and IL-8, that contribute to increase of endothelial permeability and vasodilation, and facilitate migration of inflammatory cells, mainly monocytes and neutrophils to the site of injury. MCs are capable of activating the fibroblasts and <em>keratinocytes</em>, the predominant cells involved in wound healing. MCs stimulate fibroblast proliferation during the proliferative phase via IL-4, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and basic fibroblast <em>growth</em> <em>factor</em> (bFGF) to produce a new extracellular matrix (ECM). MC-derived mediators including fibroblast <em>growth</em> <em>factor</em>-<em>2</em>, VEGF, platelet-derived <em>growth</em> <em>factor</em> (PDGF), TGF-β, nerve <em>growth</em> <em>factor</em> (NGF), IL-4, and IL-8 contribute to neoangiogenesis, fibrinogenesis, or reepithelialization during the repair process. MC activation inhibition and targeting the MC-derived mediators are potential therapeutic strategies to improve wound healing through reduced inflammatory responses and scar formation.