The repair of intestinal mucosal injuries is a tightly regulated process involving epithelial restitution, cell proliferation and maturation, and the dedifferentiation of epithelial cells. Deeper injuries also require additional repair mechanisms, including inflammatory processes, angiogenesis, and extracellular-matrix deposition. Once intestinal mucosal injury occurs, numerous <em>growth</em> <em>factors</em> and cytokines, including hepatocyte <em>growth</em> <em>factor</em> (HGF), <em>keratinocyte</em> <em>growth</em> <em>factor</em>, endothelial <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta1, intestinal trefoil <em>factor</em>, interleukin (IL)-1, and IL-<em>2</em>, are induced in both the intestinal lumen and submucosa, and these <em>factors</em> cooperatively stimulate epithelial mucosal repair. HGF, a major agent promoting hepatocyte proliferation, also modulates intestinal epithelial cell proliferation and migration, leading to the acceleration of intestinal mucosal repair. Additionally, the proteolytic activation of HGF, which is mediated by HGF activator, is essential for the regeneration of injured intestinal mucosa. Recently, several studies have shown that the administration of recombinant human HGF or HGF gene therapy abrogates disease severity in several animal models of inflammatory bowel disease (IBD). Recombinant human HGF will soon be available for administration to patients with fulminant hepatic failure. Although additional preclinical biological studies are required, HGF has the potential to be an important new treatment modality promoting intestinal mucosal repair in patients with IBD.

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of <em>2</em>,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor <em>2</em>, and transforming <em>growth</em> <em>factor</em> alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R<em>2</em>>> 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor <em>2</em> and transforming <em>growth</em> <em>factor</em> alpha mRNA, both previously shown to be induced by TCDD in human <em>keratinocytes</em>, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.

TNF-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis <em>factor</em> (TNF) family, is a multifunctional cytokine that regulate cellular proliferation, angiogenesis, inflammation, and apoptosis. In this study, we investigated the effect of TWEAK on human <em>keratinocytes</em>. Primary cultured normal human <em>keratinocytes</em> constitutively expressed a TWEAK receptor, fibroblast <em>growth</em> <em>factor</em>-inducible 14 (Fn14), and produced regulated on activation, normal T expressed and secreted (RANTES) upon TWEAK stimulation in a concentration-dependent manner. The TWEAK-induced RANTES production was abrogated by anti-Fn14 antibody, and synergistically augmented by simultaneous stimulation with transforming <em>growth</em> <em>factor</em>-beta. In addition, human <em>keratinocytes</em> differentiated in vitro with high Ca(<em>2</em>+)-containing medium showed enhanced production of RANTES upon TWEAK stimulation. Furthermore, TWEAK induced rapid phosphorylation of IkappaB-alpha in human <em>keratinocytes</em>. Collectively, TWEAK acts on human <em>keratinocytes</em> as an inducer of RANTES via Fn14. Because RANTES has been implicated in inflammation, TWEAK/Fn14 interaction in human <em>keratinocytes</em> may be involved in the pathophysiology of inflammatory skin disorders.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer <em>growth</em> and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast <em>growth</em> <em>factor</em> (FGF) receptor type II (FGFR-<em>2</em>) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-<em>2</em> in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-<em>2</em> and examined the expression of KGF, KGFR, and FGFR-<em>2</em> in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-<em>2</em> immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-<em>2</em> immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-<em>2</em> immunoreactivity. By in situ hybridization, KGF, KGFR, and FGFR-<em>2</em> were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-<em>2</em> are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell <em>growth</em> in vivo.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), a mesenchymal cell derived paracrine <em>growth</em> <em>factor</em> that regulates normal epithelial cell proliferation, appears to be an essential mediator of steroids in various reproductive organs. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human breast carcinoma tissues by immunohistochemistry. We also compared the results with the expression of estrogen receptor alpha(ERalpha), ERbeta, the proliferative activity assessed by the labeling index (LI) for the Ki-67 antigen, apoptotic frequency assessed by terminal dUTP nick end-labeling (TUNEL) index, and the expression of Bcl-<em>2</em>. All of KGF-positive cases were ERalpha- positive (p<0.05), but not that of ERbeta, while all of KGFR-positive cases were ERbeta-positive (p<0.05), but not that of ERalpha. The specimens with the coexpression of KGF and KGFR significantly correlated with a lower TUNEL index (p<0.05), but not with Ki-67 LI in breast cancer tissues. Further analysis at the cellular level revealed that Bcl-<em>2</em> was colocalized in KGFR-positive cells, and these cells were almost negative for TUNEL staining. Bcl-<em>2</em>-positive cells were also associated with ERbeta, as expected. Therefore, the results indicate that ERalpha may be involved in KGF expression, and that the coexpression of KGF and KGFR may play an inhibitory role in the induction of apoptosis possibly through the up-regulation of Bcl-<em>2</em> expression in human breast cancer.

BACKGROUND

Repifermin (<em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em>) has been shown to reduce inflammation in animal models of colitis.

OBJECTIVE

To evaluate repifermin for the treatment of active ulcerative colitis.

METHODS

Eighty-eight patients with active ulcerative colitis were enrolled in a 6-week, double-blind trial. Patients were randomized to receive treatment for five consecutive days with intravenous repifermin at a dose of 1, 5, 10, <em>2</em>5 or 50 microg/kg, or placebo. The primary objective of the study was to evaluate the safety of repifermin. The primary efficacy outcome was clinical remission at week 4, defined as a score of zero on the endoscopic appearance and stool blood components of the Mayo score and a score of zero or unity on the stool frequency and physician's global assessment components.

RESULTS

At week 4, the rates of clinical remission in the 1, 5, 10, <em>2</em>5 and 50 microg/kg repifermin groups were 19%, 9%, 0%, 0% and 0%, respectively, and 11% for the placebo group (P = 0.3<em>2</em> for repifermin vs. placebo). The frequencies of commonly occurring adverse events and severe adverse events were similar in both groups.

CONCLUSIONS

Intravenous repifermin at a dose of 1-50 microg/kg was very well tolerated, but there was no evidence that repifermin was effective for the treatment of active ulcerative colitis at these doses. An additional study to determine the efficacy of repifermin at doses of>> 50 microg/kg or for a longer treatment duration may be warranted, as the maximally tolerated dose was not reached in the present study.

The family of Neu differentiation <em>factors</em> (NDFs, or heregulins) includes a dozen secreted glycoproteins, whose receptor binding domain displays two variants, alpha and beta, and they bind to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Certain isoforms were reported to induce <em>growth</em>-arrest and differentiation of mammary tumor cells, while other breast cancer cell lines responded mitogenically. The present study addressed the biologic effects of various NDF isoforms on normal EGF-dependent epithelial cells, Balb/MK <em>keratinocytes</em>, that can undergo either proliferation or differentiation. We found that beta isoforms of NDF induced a mitogenic effect, that was significantly smaller than the maximal response to EGF. By contrast with NDF-beta, NDF-alpha isoforms exerted almost no mitogenic effect, but they were sufficient to maintain <em>keratinocytes</em> in culture. Consistent with their higher mitogenic potency, NDF-beta isoforms bound to Balb/MK cells with higher affinity (Kd = <em>2</em>.<em>2</em> nM) than alpha isoforms, however both groups shared their receptor, that we identified as ErbB-3. No transcript of ErbB-4 was detectable in the <em>keratinocytes</em>, but these cells express multiple NDF mRNAs and also ErbB-<em>2</em>. We conclude that different isoforms of NDF induce distinct <em>growth</em> regulatory effects on cultured <em>keratinocytes</em>, through direct interaction with ErbB-3.

The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM) on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various <em>growth</em> <em>factors</em> and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC-in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE)-has great merits for skin regeneration. In this study, human primary <em>keratinocytes</em> (HKs), which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.<em>2</em><em>2</em> μg/mL) than in the control group. DNA gene chip demonstrated that AAPE in <em>keratinocytes</em> (p < 0.05) notably affected expression of <em>2</em>90 identified transcripts, which were associated with cell proliferation, cycle and migration. More <em>keratinocyte</em> wound healing and migration was shown in the experimental group (1.<em>2</em><em>2</em> μg/mL). AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in <em>2</em>-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained <em>growth</em> <em>factor</em>/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-β3 differing from that shown by <em>2</em>-D analysis.

CONCLUSIONS

AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration.

The <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR)/fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>b is activated by high-affinity-specific interaction with two different ligands, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF)/fibroblast <em>growth</em> <em>factor</em> (FGF)7 and FGF10/KGF<em>2</em>, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast <em>growth</em> <em>factor</em> receptor substrate <em>2</em> (FRS<em>2</em>) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.

BACKGROUND

We have previously demonstrated that skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin (TSP)-1 prevented chronic ultraviolet (UV) B-induced angiogenesis, inflammatory cell infiltration and cutaneous photodamage in mice.

OBJECTIVE

To elucidate the mechanisms by which acute UVB-induced angiogenesis induces dermal damage, and to study the molecular regulation of acute UVB-induced angiogenesis in human skin.

METHODS

We subjected five healthy volunteers to acute UVB irradiation (<em>2</em> minimal erythema doses) and performed histological analysis at 48 h after UVB irradiation.

RESULTS

Histology revealed epidermal hyperplasia, infiltration of elastase-producing neutrophils and elastin fibre damage. Immunohistochemistry for CD31 demonstrated pronounced angiogenesis with a significant increase in both vascular density and vessel size, associated with increased endothelial cell proliferation. Whereas constitutive expression of TSP-1 but only weak expression of vascular endothelial growth factor (VEGF) were detected in normal human epidermis, pronounced downregulation of TSP-1 and upregulation of VEGF were observed in epidermal keratinocytes after acute UVB irradiation. These findings were confirmed by quantitative reverse transcription-polymerase chain reaction analysis after UVB irradiation of cultured HaCaT keratinocytes in vitro.

CONCLUSIONS

Together, these data indicate that a disruption of the balance between VEGF and TSP-1 expression leads to a UVB-induced angiogenic switch, facilitating the infiltration of elastase-producing leucocytes and cutaneous photodamage.

Arsenic causes cancer in humans, but its mechanism of action is unique among known carcinogenic agents. As a naturally occurring component of sediments and ground water, human exposure to arsenic is inevitable, necessitating the establishment of exposure limits. Because cancer is characterized as an imbalance between cell <em>growth</em> and differentiation, it has been hypothesized that arsenic exerts its carcinogenic effect, in part, by perturbing the balance between these antagonistic processes. Previous work in this laboratory has demonstrated that sodium arsenite prevents adipocytic differentiation of C3H 10T1/<em>2</em> cells, leading to the hypothesis that the underlying mechanism involves downregulation of genes associated with adipogenesis. In support of this hypothesis, it was found that mRNA levels of peroxisome proliferative-activated receptor gamma (PPAR gamma), CCAAT-enhancer binding protein alpha (C/EBP alpha), and adipocyte-selective, fatty acid-binding protein (aP<em>2</em>) are decreased in arsenic-treated cells; arsenic-induced phenotypic reversion of differentiated adipocytes correlates with reduced aP<em>2</em> expression. Arsenic also blocks upregulation of p<em>2</em>1(Cip1/Waf1), a <em>factor</em> whose expression is tightly regulated during adipogenesis. The differentiating effect of pioglitazone, which induces adipogenesis by activating PPAR gamma, is inhibited by arsenic, suggesting that arsenic interferes with adipogenic signaling at or below the level of PPAR gamma. Because C/EBP alpha is important in the expression of certain <em>keratinocyte</em>-specific genes, the negative effect of arsenic on C/EBP alpha might also contribute to the development of skin cancer. PPAR gamma, C/EBP alpha, and p<em>2</em>1(Cip1/Waf1) are important in numerous normal and pathological processes, including carcinogenesis, leading us to postulate that perturbation of these <em>factors</em> by arsenic might contribute to the carcinogenic effect of this metalloid.

BACKGROUND

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>) also described as fibroblast <em>growth</em> <em>factor</em>-10 (FGF-10) is a newly identified member of the fibroblast <em>growth</em> <em>factor</em> family. KGF-<em>2</em> is 96% identical to the recently identified rat FGF-10 and specifically stimulates <em>growth</em> of normal human epidermal <em>keratinocytes</em>. The present study was undertaken to examine the effects of topically applied KGF-<em>2</em> in an incisional wound healing model. KGF-<em>2</em> treatment resulted in an improvement in incisional wound healing as characterized by an increase in breaking strength, collagen content, and epidermal thickness.

METHODS

KGF-<em>2</em> was topically applied to linear incisions made in the dorsal skin of Sprague-Dawley rats. Biomechanical testing was done using an Instron tensiometer for breaking and tensile strength determinations. Wound collagen content was determined using the Sircol collagen assay. Epidermal thickness measurements were conducted using Masson's trichrome-stained sections of the wound.

RESULTS

A single topical application of KGF-<em>2</em> at the time of wounding resulted in an increase in wound breaking and tensile strength at Day 5 after wounding. Breaking strength of KGF-<em>2</em>-treated wounds was significantly higher compared with the buffer control (1 microgram, <em>2</em><em>2</em><em>2</em>.1 +/- 13.5 g, P = 0.0007; 4 microgram, <em>2</em>48.7 +/- 15.4 g, P = 0.0001; 10 microgram, <em>2</em>47.<em>2</em> +/- <em>2</em>1.9 g, P = 0.001; buffer, 141.0 +/- 9.7 g). Epidermal thickness and wound collagen content were significantly increased following treatment with KGF-<em>2</em>.

CONCLUSIONS

Based on our findings, KGF-<em>2</em> is a potent stimulator of wound healing as demonstrated by increased mechanical strength accompanied by an increase in wound collagen content. KGF-<em>2</em> could be an important cellular mediator responsible for the initiation and acceleration of wound healing and may enhance the healing of surgical wounds.

BACKGROUND

Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization).

RESULTS

Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and <em>keratinocytes</em>, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/<em>2</em>) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and <em>growth</em> <em>factors</em>. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and <em>2</em>0% v/v). Whereas both platelet lysate concentrations increased cell migration, only <em>2</em>0% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/<em>2</em> and NFκB with the same early kinetics, whereas the effect was different for later time-points.

CONCLUSIONS

These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Large-surface or deep wounds often become senescent in the inflammatory or proliferation stages and cannot progress to reepithelialization. This failure makes intervention necessary to provide the final sealing epithelial layer. The best current treatment is autologous skin graft, although there are other choices such as allogenic or autologous skin substitutes and synthetic dressings. Amniotic membrane (AM) is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial reepithelialization effects, with antiinflammatory, antifibrotic, antimicrobial, and nontumorigenic properties. These properties are related to its capacity to synthesize and release cytokines and <em>growth</em> <em>factors</em>. We report the use of AM as a wound dressing in two patients with large and deep traumatic wounds. Negative pressure wound therapy followed by AM application was capable of restoring skin integrity avoiding the need for skin graft reconstruction. AM induced the formation of a well-structured epidermis. To understand this effect, we designed some assays on human <em>keratinocyte</em>-derived HaCaT cells. AM treatment of HaCaT induced ERK1/<em>2</em> and SAP/JNK kinases phosphorylation and c-jun expression, a gene critical for <em>keratinocytes</em> migration; however, it did not affect cell cycle distribution. These data suggest that AM substantially modifies the behavior of <em>keratinocytes</em> in chronic wounds, thereby allowing effective reepithelialization.

The urokinase receptor (uPAR) is involved in a series of pathological processes, from inflammation to cancer. We have analyzed in detail the role of uPAR and the mechanisms involved in <em>keratinocyte</em> behavior during wound healing by exploiting uPAR-knockout (KO) mice. In vivo, uPAR-KO mice showed delayed wound healing, with abnormal <em>keratinocyte</em> migration and proliferation. In vitro, unlike wild-type cells, primary uPAR-KO <em>keratinocytes</em> did not proliferate in response to epidermal <em>growth</em> <em>factor</em> (EGF), their <em>growth</em> and migration were not inhibited by EGF-receptor (EGFR) inhibitors, and they did not adhere to uncoated surfaces. Whereas EGFR levels in uPAR-KO <em>keratinocytes</em> were normal, there was no tyrosine phosphorylation upon addition of EGF, and its downstream targets, extracellular-signal-regulated kinases 1 and <em>2</em> (ERK1/<em>2</em>), were not activated. Re-introduction of mouse uPAR rescued all phenotypes. In vitro adhesion and migration defects were associated with the failure of uPAR-KO <em>keratinocytes</em> to normally produce and secrete laminin-5 (LN5), an event that requires EGFR signaling. These results were confirmed in vivo, with LN5 being upregulated during wound healing in wild-type but not in uPAR-KO epidermis.

Epithelial-mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal <em>keratinocytes</em> assessed by immunocytochemistry and profiling of gene activation for <em>growth</em> <em>factors</em>/cytokines by microarray chip technology. IGF-<em>2</em> and BMP-4 were determined as candidate <em>factors</em> responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-<em>2</em> and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these <em>growth</em> <em>factors</em> was also detected in tumor samples.

Narrow-band ultraviolet-B (UVB) radiation is an effective treatment for vitiligo vulgaris. However, the mechanisms of narrow-band UVB in inducing repigmentation of vitiligo lesions are not thoroughly clarified. The purpose of our study was to investigate the effects of narrow-band UVB irradiation on melanocyte proliferation and migration in vitro. Our results showed that the cell counts as well as [3H]thymidine uptake of melanocytes were significantly enhanced by narrow-band UVB-irradiated <em>keratinocyte</em> supernatants. In these supernatants, a significant increase in basic fibroblast <em>growth</em> <em>factor</em> (bFGF) and in endothelin-1 (ET-1) release was observed. bFGF is a natural mitogen for melanocytes, whereas ET-1 can stimulate DNA synthesis in melanocytes. This stimulatory effect of melanocyte proliferation by supernatants derived from narrow-band UVB-irradiated <em>keratinocytes</em> was significantly reduced by a selective endothelin-B (ET-B) receptor antagonist (BQ788), suggesting an essential role of ET-1 on melanocyte proliferation. Our results of time-lapse microphotography revealed a stimulatory effect of narrow-band UVB irradiation on melanocyte migration. Focal adhesion kinase (FAK) plays a pivotal role in cell migration. Phosphorylated FAK (p1<em>2</em>5(FAK)) expression on melanocyte was enhanced by narrow-band UVB irradiation. In this study, narrow-band UVB irradiation stimulated a significant increase in matrix metalloproteinase-<em>2</em> (MMP-<em>2</em>) activity in melanocyte supernatants. Narrow-band UVB-irradiation-induced migration of melanocytes was significantly annihilated by the addition of p1<em>2</em>5(FAK) inhibitor (herbimycin-A) or MMP-<em>2</em> inhibitor (GM6001). These results suggest that p1<em>2</em>5(FAK) and MMP-<em>2</em> activity play important roles in narrow-band UVB-induced migration of melanocytes. Our results provide a theoretical basis for the effectiveness of narrow-band UVB irradiation in treating vitiligo.

We evaluated the effects of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha <em>2</em>-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) causes a proliferation of pancreatic ductal epithelial cells in adult rats after daily systemic administration for 1 to <em>2</em> weeks. Even before the proliferation of intralobular ducts is histologically evident, KGF also induces proliferating cell nuclear antigen expression within the ductal epithelium of intercalated, intralobular, and interlobular ducts. KGF also causes incorporation of 5-bromodeoxyuridine in ductal epithelial cells. Epithelial cell proliferation is histologically most prominent at the level of the intralobular ducts adjacent to and within the islets of Langerhans. Pancreatic ductal proliferation is not histologically apparent in rats sacrificed 7 to 10 days after the cessation of KGF administration. The pancreatic hormones insulin, glucagon, somatostatin, and pancreatic polypeptide are normally distributed within islets that demonstrate intrainsular ductal proliferation. The proliferating ductal epithelium does not show endocrine differentiation as evidenced by the lack of immunoreactivity for pancreatic hormones. KGF is a potent in vivo mitogen for pancreatic ductal epithelial cells.

The majority of biological responses classically attributed to tumor necrosis <em>factor</em> alpha (TNF-alpha) is mediated by p55 receptor (TNFR1). Here, we aimed to clarify the biological role of TNFR1-mediated signals in an in vivo inflammatory angiogenesis model. Polyester-polyurethane sponges, used as a framework for tissue <em>growth</em>, were implanted in C57Bl/6 mice. These implants were collected at days 1, 7, and 14 post-implant for enzyme-linked immunosorbent assay or at days 7 and 14 for hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, used as indexes for angiogenesis, neutrophil, and macrophage accumulation, respectively. In TNFR1-deficient C57Bl/6 mice, there was a significant decrease in sponge vascularization but not in late inflammatory cell influx. It is interesting that levels of vascular endothelial <em>growth</em> <em>factor</em> were significantly lower in TNFR1-deficient than in wild-type mice at days 1 and 7. Levels of angiogenic chemokines, CC chemokine ligand <em>2</em>/murine homologue of monocyte chemoattractant protein-1 and CXC chemokine ligand 1-3/<em>keratinocyte</em>-derived chemokine, were significantly lower in TNFR1-deficient mice at days 1 and 7 after implantation, respectively. These observations suggest that TNFR1-mediated signals have a critical role in sponge-induced angiogenesis, possibly by influencing the effector state of inflammatory cells and hence, modulating the angiogenic molecular network.

BACKGROUND

Surgical and traumatic injuries to the bladder initiate a complex series of biological processes that result in wound healing. This process involves cellular proliferation, migration and differentiation; removal of damaged tissue; and production of extracellular matrix all of which may be controlled by growth factors. In skin, keratinocyte growth factor (KGF) is induced following incisional injury. We hypothesize that in bladder wound healing KGF and other growth factors are induced to modulate tissue repair.

METHODS

We have created a model of surgical bladder injury in the rodent. At 12, 24 and 48 hrs and 5 and 7 days after injury, the bladder was bisected and total RNA extracted from the anterior or wounded half and posterior or non-wounded half. Histological analysis of the bladder wound was performed with Mason's Trichrome and immunohistochemistry against smooth muscle alpha actin. RNase protection assays were performed to examine the expression of KGF, transforming growth factor (TGF)alpha and TGF beta 2 and 3 as well as the receptors for KGF and epidermal growth factor (EGF). Lastly, the effects of the exogenous administration of KGF on the bladder was tested on neonatal mice by daily injections of 5 micrograms KGF per gram body weight for 5 days.

RESULTS

At 12 hours after injury KGF mRNA expression in the anterior wounded bladder half and posterior non-wounded bladder half was 8 and 6 times higher respectively, compared to unoperated control bladders. A similar response was seen for TGF alpha, where the 12 hour mRNA expression was 4.5 times higher in the anterior wounded bladder half and 3.5 times higher in the posterior non-wounded bladder half compared to unoperated control bladders. The nadir mRNA expression for both KGF and TGF alpha occurred at 7 days after bladder injury and was the same as in unoperated control bladders. EGFR mRNA expression was approximately 2 times higher in both the anterior wounded and posterior non-wounded bladder halves compared to the nadir levels which occurred at 24 hours after injury. TGF beta 2 and beta 3 mRNA levels did not significantly change in either the anterior wounded or posterior non-wounded bladder halves. Exogenous KGF stimulation resulted in a marked urothelial proliferation when compared to age matched control animals.

CONCLUSIONS

During the early phases of bladder wound healing (12-24 hours post injury), mRNA for KGF and TGF alpha increased, whereas TGF beta 2 and beta 3 and the KGFR and EGFR remain unchanged. Additionally, exogenous KGF has a direct effect on urothelial proliferation. KGF and TGF alpha warrant further study as potential mediators of bladder wound healing.

Aging is the highest risk <em>factor</em> for cancer. Although oxidants are thought to contribute to both aging and cancer, the interplay between oxidative stress, aging, and cancer has not been well studied. Human diploid fibroblasts (HDFs) undergo premature senescence in response to sublethal doses of H(<em>2</em>)O(<em>2</em>). To test the hypothesis that senescent or senescent-like HDFs function as a tumor promoter, we have employed an in vitro skin tumor promotion model, in which colony formation is measured using initiated mouse <em>keratinocyte</em> 308 cells seeded at clonal density. 308 cells form colonies when co-cultured with normal HDFs only in the presence of the tumor promoter phorbol 1<em>2</em>-myristate 13-acetate (TPA), which induces an average of 5.75 colonies. When co-cultured with H(<em>2</em>)O(<em>2</em>)-treated HDFs, 308 cells form an average of 30.3 colonies. To understand the mechanism behind this phenomenon, we tested whether conditioned medium of HDFs, HDF extracellular matrix (ECM), density of HDFs, or the contact between <em>keratinocytes</em> and HDFs plays a role in 308 cell colony formation. The conditioned medium from prematurely senescent cells resulted in an average of eightfold more 308 cell colonies formed than the conditioned medium from normal HDFs, and the <em>growth</em>-promoting effect of the conditioned medium was trypsin sensitive. The ECM alone was not able to induce 308 cell colony formation. Increasing the density of normal HDFs or contact with normal HDFs but not senescent-like HDFs was inhibitory to the <em>growth</em> of 308 cells. Measurement of Connexin 43 indicated a decreased expression of the protein, which suggests an impaired gap junction communication in senescent-like HDFs. We conclude that H(<em>2</em>)O(<em>2</em>)-treated fibroblasts not only lose contact inhibition of the <em>growth</em> of initiated <em>keratinocytes</em> perhaps related to reduced gap junction communication but also increase production of secreted protein <em>factors</em> to enhance the <em>growth</em> of 308 <em>keratinocytes</em>.

Abnormalities in the expression and signaling pathways downstream of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) contribute to malignant transformation in human cancers, including those of the cutaneous epithelium. Accordingly, novel agents such as the EGFR tyrosine kinase inhibitor ZD1839 (Iressa), are promising, biologically based treatments that are currently in preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival. This study explored the effect of ZD1839 on the stimulation of p4<em>2</em>/44 mitogen-activated protein kinase (MAPK) and p<em>2</em>1-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival, using immortalized <em>keratinocytes</em> (HaCaT cells) and cutaneous squamous cell carcinoma cells. The EGFR and a number of effector kinases (mitogen-activated protein extracellular signal-regulated kinase kinase 1 and <em>2</em>, MAPK, Pak1, p38, c-JunNH(<em>2</em>)-terminal kinase and extracellular signal-regulated kinase 1) and cell survival proteins (AKT, FKHR, and c-Src) showed constitutive pathway activation in HaCaT and cutaneous squamous cell carcinoma cells. ZD1839 effectively inhibited EGFR and MAPK activation and Pak1 activity in exponentially <em>growing</em> cancer cells. ZD1839 also suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Y1068, MAPK phosphorylation on T40<em>2</em> and Y404, and Pak1 activity in a dose-dependent manner. In addition, ZD1839 blocked EGF-induced cytoskeleton remodeling, cell <em>growth</em>, and in vitro invasiveness of cancer cells and induced a differentiated squamous cell phenotype. These studies suggest that the EGFR-tyrosine kinase inhibitor ZD1839 may cause potent inhibition of the EGFR, MAPK, and Pak1 pathways, resulting in attenuation of transformed cell phenotypes and induced differentiation in human cancer cells deregulated in these <em>growth</em> <em>factor</em> receptor pathways.

Betulinic acid (BA), a pentacyclic triterpene first identified less than a decade ago, has served as a melanoma-specific cytotoxic agent, and yet its specificity is being challenged. Recently, we found that human melanoma cells exhibited less sensitivity to betulinic acid than human skin <em>keratinocytes</em>. This study was designed to investigate the cell signaling pathway leading human melanoma cells to increased resistance to betulinic acid treatment. In vitro experiments using cultured human melanoma cells indicated that betulinic acid transiently induced survivin expression. The expression of survivin started 30 min post-betulinic acid treatment, peaked at <em>2</em> h, remained elevated for 8 h and returned to basal level within <em>2</em>4 h. Similarly, epithelial <em>growth</em> <em>factor</em> (EGF) treatment induced expression of survivin in a time-dependent manner. Since epithelial <em>growth</em> <em>factor</em> receptor (EGFR) activation leads to the activation of cell signaling components that are important to cell survival, we next examined whether BA-induced survivin expression is mediated by the EGFR pathway. The results showed that BA induced EGFR tyrosine phosphorylation in a time-dependent manner. Further, BA strongly induced AKT phosphorylation in a similar pattern. AKT activation started 15 min post-treatment, peaked at approximately 1 h, remained elevated for 4 h and returned to basal level within 8 h. BA also induced ERK activation and, in contrast, weakly induced JNK and p38 activation. Pretreatment of EGFR inhibitor PD153035 blocked BA-induced EGFR phosphorylation, ERK and AKT activation, and survivin expression. Results of the MTT dye assay showed that a combination of PD153035 and BA enhanced melanoma cell death. Collectively, we conclude that betulinic acid transiently activated the EGFR/AKT cell survival pathway and induced survivin expression, contributing to less sensitivity in human melanoma cells. The data suggest that a combination of the EGFR inhibitor and betulinic acid may be a better clinical option to treat human melanoma.