Keratinocyte Growth Factor Receptor Research Articles

KGF Prevents Oxygen-Mediated Damage in ARPE-19 Cells

Oxidative stress has been implicated in a variety of diseases of the eye. In several other tissues, keratinocyte growth factor (KGF) has been shown to prevent negative cellular changes associated with oxidative insult, such as permeability increases and nuclear DNA damage. In this study, we looked at whether KGF provided these same protective effects to cultured human retinal pigmented epithelial (RPE) cells (ARPE-19). Reverse transcriptase-polymerase chain reaction (RT-PCR) using a published primer pair sequence followed by restriction endonuclease digestion with AvaI and HincII was used to look for the KGF receptor message in ARPE-19 cells. Cellular response to KGF was verified through proliferation assays and Western blot analysis for mitogen-activated protein kinase (MAPK). Single-cell gel electrophoresis was used to assess DNA damage, Western blot analysis was used to assay actin cytoskeletal changes, and electrical resistance and tracer experiments with Transwell tissue plates were used to assess permeability changes. Immunostaining was used to verify the existence of the tight junction protein occludin. It was verified through RT-PCR that the ARPE-19 cell line exhibited the message for FGFR2-IIIb, otherwise known as KGFR. KGF was also shown to increase cellular proliferation and activated the MAPK p44/p42 cascade. KGF ameliorated nuclear DNA damage and cytoskeletal rearrangement caused by oxidative stress through the addition of exogenous hydrogen peroxide but was unable to prevent permeability changes. KGF was shown to significantly reduce DNA damage and cytoskeletal rearrangement caused by oxidative stress in cultured ARPE-19 cells. This result may be useful in targeting future therapies to combat a multitude of diseases of the eye that result from increases in reactive oxygen species.

RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation

The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.

Cell Death-Induced Activation of Epidermal Growth Factor Receptor in Keratinocytes: Implications for Restricting Epidermal Damage in Dermatitis

Recent findings have implicated Fas/Fas ligand (FasL) in mediating the death of keratinocytes in spongiotic lesions. We asked whether dying keratinocytes could potentially initiate a protective response of the skin to limit the destruction of the epidermis in the spongiotic areas. In addition to apoptosis, treatment of keratinocyte cultures in vitro with FasL triggers a profound phoshorylation of the epidermal growth factor receptor (EGFR) and of its downstream effectors ERK and protein kinase B (PKB/Akt). Using a variety of inhibitors and blocking antibodies, we demonstrated that: (i) apoptosis is required for the generation of the signal(s) leading to the activation of EGFR, ERK, and Akt; (ii) the activation of EGFR, ERK, and Akt by FasL is indeed mediated by its bona fide receptor Fas; (iii) the activation of EGFR is essential for the subsequent activation of ERK and Akt; and (iv) apoptotic keratinocytes secrete soluble EGFR ligands (including amphiregulin) that are processed from membrane-bound proligand forms by metalloproteinase(s). Our findings demonstrate a potential mechanism for the restriction and repair of spongiotic damage in eczemas.

Esophageal ATP synthase and keratinocyte growth factor gene expression changes after acid and bile-induced mucosal damage

Intramural gene expression changes may be critically involved in tissue damage, defense and repair after esophageal regurgitation. The aims were to characterize the consequences of short-term exposure to luminal bile, acid, or bile mixed with acid on the beta-ATPase, keratinocyte growth factor 1 (KGF-1) and KGF receptor (KGF-R) expressions within the mucosa and the muscle layer in a large animal model. Esophageal segments of anesthetized dogs were exposed to saline (n = 3), diluted canine bile (n = 6), hydrochloric acid (n = 5) or bile + hydrochloric acid (n = 5), and tissue biopsies were taken at the end of the 180-min observation period. Semiquantitative reverse transcriptase polymerase chain reactions were carried out and the degree of histological damage was evaluated on the 0-16-grade Geisinger scoring scale. Acid exposure was followed by a significant decrease in the level of beta-ATPase expression in the mucosa, and parallel increases in KGF-1 and KGF-R expression. Corresponding changes in the muscle layer were not significant. Bile alone evoked more severe tissue damage, with significantly decreased beta-ATPase levels in both the mucosa and the muscle, whereas the KGF-1 expression did not change significantly. The bile + acid treatment induced an intermediate state, with significant beta-ATPase transcription level decreases in both layers, while the mucosal KGF-1 expression was lower than that following acid treatment alone. The acid-induced transcriptional level downregulation of mucosal beta-ATPase gene expression in the smooth muscle layer was exacerbated by bile, but the concomitant KGF and KGF-R gene expression changes may indicate the start of a consecutive repair process.

Gene expression profiling identifies activating transcription factor 3 as a novel contributor to the proapoptotic effect of curcumin

The antitumor effect of curcumin (diferuloylmethane) is well established. However, there have been no unbiased studies to identify novel molecular targets of this compound. We therefore undertook a gene expression profiling study to identify novel targets of curcumin. A cDNA array comprised of 12,625 probes was used to compare total RNA extracted from curcumin-treated and untreated MDA-1986 cells for differential gene expression. We identified 202 up-regulated mRNAs and 505 transcripts decreased > or =2-fold. The proapoptotic activating transcription factor 3 (ATF3) was induced >4-fold. Two negative regulators of growth control [antagonizer of myc transcriptional activity (Mad) and p27kip1] were induced 68- and 3-fold, respectively. Additionally, two dual-activity phosphatases (CL 100 and MKP-5), which inactivate the c-jun-NH2-kinases, showed augmented expression, coinciding with reduced expression of the upstream activators of c-jun-NH2-kinase (MEKK and MKK4). Of the repressed genes, the expression of Frizzled-1 (Wnt receptor) was most strongly attenuated (8-fold). Additionally, two genes implicated in growth control (K-sam, encoding the keratinocyte growth factor receptor, and HER3) as well as the E2F-5 transcription factor, which regulates genes controlling cell proliferation, also showed down-regulated expression. Considering its role in apoptosis, we determined the contribution of ATF3 to the antitumor effect of curcumin. Curcumin-treated MDA-1986 cells showed a rapid, dose-dependent increase in ATF3/mRNA protein. Moreover, expression of an exogenous ATF3 cDNA synergized with curcumin in inducing apoptosis. Thus, we have identified several putative, novel molecular targets of curcumin and showed that one, (ATF3) contributes to the proapoptotic effects of this compound.

Keratinocyte growth factor‐1 expression in healthy and diseased human periodontal tissues

Keratinocyte growth factor-1 (KGF-1) is up-regulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific keratinocyte growth factor receptor (KGFR). We examined KGF-1 and KGFR protein and gene expression in healthy and diseased periodontal tissues. Tissues were collected from patients with periodontal health or disease, immediately frozen and stained for KGF-1 and KGFR protein expression. Laser capture microdissection of epithelial and connective tissue cells with reverse transcription-polymerase chain reaction (RT-PCR) examined KGF-1 and KGFR gene expression profiles and enzymatic digestion with heparitinase, chondroitinase ABC or pre-treatment with suramin examined epithelial surface molecule interactions with KGF-1. In tissues collected from healthy patients, KGF-1 protein localized to areas of junctional and basal oral epithelial cells and was significantly increased in periodontal pocket epithelium (p<0.01) and in the oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, with the relative staining intensity being increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmed KGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. KGF-1 localization to epithelial cells was largely eliminated by suramin pre-treatment, indicating interaction with the KGFR. KGF-1 and KGFR proteins are expressed in healthy periodontal tissues but significantly increased in diseased periodontal tissues. We hypothesize up-regulation of KGF-1 and KGFR protein associated with disease regulates epithelial cell behavior associated with onset and progression of periodontal pocket formation.

Activation of the keratinocyte growth factor receptor is important to control cell cycle progression after partial hepatectomy: Torsten Wuestefeld, Medical School Hannover, Hannover Germany; Heike Steiling, ETH, Zuerich Switzerland; Stefan Kubicka, Medical School Hannover, Hannover Germany; Jeffrey I Gordon, Washington University, St Louis, Michael P Manns, Medical School Hannover, Hannover Germany; Sabine Werner, ETH, Zuerich Switzerland; Christian Trautwein, Medical School

Activation of the keratinocyte growth factor receptor is important to control cell cycle progression after partial hepatectomy: Torsten Wuestefeld, Medical School Hannover, Hannover Germany; Heike Steiling, ETH, Zuerich Switzerland; Stefan Kubicka, Medical School Hannover, Hannover Germany; Jeffrey I Gordon, Washington University, St Louis, Michael P Manns, Medical School Hannover, Hannover Germany; Sabine Werner, ETH, Zuerich Switzerland; Christian Trautwein, Medical School

Expression of keratinocyte growth factor and its receptor in adaptive changes of ileorectal pouch mucosa

Objective: Total proctocolectomy with formation of an ileo-anal pouch is a well-established surgical procedure for patients with ulcerative colitis (UC) or familiar adenomatous polyposis (FAP). The pouch mucosa undergoes adaptive changes, with inflammation of the ileal reservoir being the most common complication. The aetiology is unknown. The keratinocyte growth factor (KGF) has not only been shown to promote intestinal wound healing and re-epithelialization but also to have some immunomodulatory properties. This study was designed to investigate a putative involvement of KGF in observed histomorphological changes in the pouch mucosa. Material and methods. Multiple biopsies were obtained from age-matched and sex-matched patients. Biopsies were stained with H&E and scored for inflammation and morphological changes. mRNA expression levels of KGF and KGF-receptor (KGFR) were determined using competitive RT-PCR, protein expression and phosphorylation was analyzed by Western blotting. KGF and KGFR were localized in tissue samples by immunohistochemistry. Results. Expression of KGF and KGFR was significantly increased in inflamed and adapting mucosa. Patterns of expression did not significantly differ in pouch mucosa from UC or FAP patients. Protein expression correlated with the mRNA results and KGFR was shown to be activated in adapting pouch mucosa. KGF was detected on subepithelial cells, mainly on fibroblasts, whereas expression of KGFR was restricted to epithelial cells. Conclusion. Expression of KGF and KGFR is significantly increased in the pouch mucosa, suggesting an involvement of this growth factor in tissue repair and adaptive changes. Topical application of KGF might alleviate the inflammatory and promote the adaptive process in the ileo-anal pouch mucosa.

Involvement of KGF in the Wound Healing Process of Tracheal Cartilage in Rat Longitudinal Tracheotomy Model

Aim: To address whether KGF (Keratinocyte Growth Factor) and KGFR (keratinocyte Growth Factor Receptor) expression is involved in the wound healing process of tracheal cartilage in rat longitudinal tracheotomy model. And to expose the new strategy to control the process utilizing KGF and its related compounds.Method: In this study, we incised rat trachea longitudinally for three cartilage lings, and harvested trachea from rat on 0, 1, 3, 5, 7 days after operation. We made horizontal section and observe them with immunohistochemistry.Result: The cartilage defect was filled with chondrocytes originated from the perichondrium. KGF expression was observed in chondrocytes, perichondrium and fibroblasts in injured area. The expression of KGF was upregulated immediately after the operation, reaching a peak at 3–5 days.Future approach: Further examination on temporal and spatial pattern of KGF and KGFR expression. Quantitative analysis of the expression of KGF and KGFR in relation to cell kinetics. Attempts to manipulate the kinetics of wound healing using KGF and its related compounds.

Protection of Epithelial Cells by Keratincoyte Growth Factor Signaling

Oxidative injury to the lung is associated with widespread injury to the alveolar epithelium, which can be fatal unless the process is controlled and repaired. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, has been shown to protect the lung from a variety of oxidative insults. The mechanism(s) underlying the protective effects of KGF in lung injury is being investigated in many laboratories. Although KGF has potent mitogenic effects on epithelial cells, the proliferative effect of KGF was shown to be abolished in oxygen-breathing animals, but KGF was still able to inhibit alveolar damage. This demonstrates that the protective effect of KGF cannot simply be explained by the ability of KGF to stimulate type II cell proliferation. To identify the mechanisms involved in the protective effects of KGF, we used an inducible lung-specific transgenic approach to overexpress KGF in murine lungs, since constitutive overexpression of KGF in the mouse affects lung development. The transgenic system allowed us to identify the pro-survival Akt pathway as an important mediator of the protective effects of KGF both in vivo and in vitro. In addition, use of a yeast two-hybrid system led to the identification two proteins p90RSK and PAK4 that associate with the KGF receptor and are important for the protective functions of KGF. Experiments are underway to determine whether the different pathways triggered by KGF all converge on the Akt pathway, or whether they independently induce protective mechanisms that along with Akt are crucial for cell survival.

Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.

Involvement of keratinocyte growth factor (KGF)-KGF receptor signaling in developmental estrogenization syndrome of mouse vagina.

Exposure of mice to estrogen or keratinocyte growth factor (KGF) in vivo during the neonatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. Here, whether and how KGF-signaling is involved in the effects of estrogen on the neonatal mouse vagina were studied with an in vitro method. Newborn mouse vaginae were cultured for 3 days in serum-free medium containing various combinations of estradiol-17beta (E2), KGF, anti-KGF antibody, KGFR inhibitory peptide and heparin, and then transplanted into ovariectomized host mice for 35 days. The vaginae cultured with 5 microg/ml E2 or 5 microg/ml KGF had a cornified thick epithelium, while the epithelium of the vehicle-treated controls stayed thin. The E2 effect was blocked by concurrent treatment with anti-KGF antibody or KGFR inhibitory peptide. KGF treatment alone at doses less than 500 ng/ml did not induce permanent vaginal changes but such changes did occur in vaginae treated with heparin plus as little as 10 ng/ml KGF. On the other hand, heparin inhibited the permanent vaginal changes induced by estrogen. These results suggest that irreversible vaginal changes are induced by the direct action of KGF on the developing vagina and that the developmental estrogenization syndrome of mouse vagina is caused by intensification of endogenous KGF/KGFR signaling by exogenous estrogen.

Activated Hepatic Stellate Cells Express Keratinocyte Growth Factor in Chronic Liver Disease

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a specific mitogen for different types of epithelial cells and a potent survival factor for these cells under stress conditions. KGF expression increases strongly after injury to various tissues, including the skin and the intestine, and signaling via the KGF receptor was shown to be crucial for repair of skin wounds and for liver regeneration. Here we demonstrate an increased expression of KGF in chronic liver disease associated with fibrosis. The extent of KGF overexpression correlated strongly with the stage of fibrosis. As the cellular source of KGF we identified activated hepatic stellate cells (HSCs)/myofibroblasts. In contrast to the ligand, the KGF receptor, FGFR2-IIIb, was exclusively expressed by hepatocytes, but not by activated HSCs or other parenchymal or nonparenchymal liver cells. Based on the known effects of KGF on hepatocytes in vitro, our findings suggest that HSC/myofibroblast-derived KGF may enhance liver regeneration and/or hepatocyte survival in patients with chronic liver disease.

Influence of keratinocyte growth factor on clonal growth of epithelial tumor cells, lymphoma and leukemia cells and on sensitivity of tumor cells towards 5-fluorouracil in vitro.

RhKGF is developed for prevention and treatment of chemotherapy- and radiation-induced epithelial damage in cancer patients. Little information exists on growth modulation of malignant cells by KGF. We have tested the anchorage-independent clonal growth of 35 human tumor and 22 lymphoma and leukemia cell lines in semisolid media with or without rhKGF. Growth of the majority of cell lines was not significantly influenced by rhKGF. Growth of 5 cell lines (2 lung, 1 stomach, 1 colorectal, 1 breast) was significantly stimulated by rhKGF. The effect was dose-dependent with significant stimulation at concentrations >1-10 ng/ml. Growth modulation was amplified by serum reduction and abolished by anti-hKGF antibodies. Responding cell lines expressed KGF receptor RNA and showed specific KGF-binding. To determine whether KGF-induced higher colony numbers represented cell divisions with resulting differentiation and growth arrest or self-renewal we performed experiments on serial plating efficacy of colony-derived tumor cells with or without continued presence of rhKGF. Results revealed KGF stimulation of colony formation as representing increase of cellular self-renewal. To test possible interaction of rhKGF with activity of cytotoxic drugs in clinical protocols, we have used combination protocols with 5-fluorouracil (5-FU). Results showed that rhKGF incubation before, after (or both) addition of 5-FU did not diminish the sensitivity of tumor cells to 5-FU cytotoxicity under serum concentrations relevant for the clinical situation. In conclusion, some epithelial tumor cells have receptors for KGF signaling for clonal growth. When considered in the planning of treatment protocols, this effect is unlikely to compromise chemotherapy sensitivity.

P0871 THE ROLE OF KERATINOCYTE GROWTH FACTOR (KGF) IN THE PATHOGENESIS OF HELICOBACTER PYLORI (HP) ASSOCIATED GASTRIC DISEASE

Introduction: Helicobacter pylori (HP) causes gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer. HP related gastric cancer develops through sequential and progressive changes in the gastric epithelial cells. Although it is thought that HP may act by affecting growth factors functions, very little is known about the identity of these growth factors and the molecular mechanisms that mediate these changes. KGF is a member of the heparin binding fibroblast growth factors (FGFs) family. KGF acts specifically on epithelial cells to elicit a variety of responses such as cell proliferation, morphogenesis and wound repair. Both, KGF and its receptor are strongly implicated in the malignant process: The KGF receptor (KGFR) was identified as an amplified gene in human stomach cancer and, overexpression of the receptor in cultured cells induces malignant transformation of the cells in a ligand-dependent manner. KGF also plays an important role in maintenance, renewal and repair of epithelial cells in the GIT. We hypothesized that KGF might be one of the major growth factors whose normal function might be affected by HP. Methods: We have comparatively defined the expression of KGF in a variety of gastric pathologies including gastritis, atrophic gastritis and gastric cancer. The validity of the immunohisto-chemistry (IHC) staining was ascertained mainly by preliminary IHC staining of pancreatic tumor samples known to express KGF, usage of a standardized IHC staining agent, Vimentin, as positive control, and by the usage of blocking antibodies with variable degrees of specificity. Results: KGF expression was observed in most gastric pathologies, especially inflammatory but also neoplastic (table). No definite correlation was found between the degree of KGF expression and HP etiological involvementTable 1: KGF localization stain according to diagnosisConclusion: KGF, according to our findings, may serve as a rather sensitive biological marker for gastric pathologies but further research is obviously needed

Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in human uterine cervical cancer

Keratinocyte growth factor receptor (KGFR), also known as FGFR2 IIIb, is a splice variant of FGFR-2. KGFR is expressed in many types of epithelial cell and is activated with four known ligands [FGF-1, FGF-3, FGF-7 (also known as KGF) and FGF-10] that are predominantly synthesized by mesenchymal cells. KGFR is highly expressed in the late-proliferative phase of a normal endometrium and in endometrial adenocarcinoma. In the present study, we attempted to determine the expression and localization of KGFR in human cervical cancer cell lines and cervical cancer tissues. The KGFR protein was detected in CaSki and HeLa cells, but not in ME-180 cells of cervical cancer cell lines. In non-cancer cervical tissues, KGFR immunoreactivity was weakly expressed in the surface of squamous epithelial cells and vascular smooth muscle cells. Immunohistochemically, the KGFR protein was detected in squamous cell carcinoma in 36 of 42 (86%) cervical cancer patients. In cervical cancer tissues, KGFR was detected in 17 of 18 (94%) of patients with the keratinizing type and 19 of 24 (79%) of patients with the non-keratinizing type of cervical cancer. Furthermore, KGFR was prominently localized in proliferating reserve cells and squamous metaplastic reserve cells adjacent to cancer cells. In contrast, KGFR was not detected in cervical ductal cells in cancer or non-cancer cervical tissues. These findings may indicate that KGFR mediates the growth and differentiation of reserve cells and squamous cell carcinoma in the cervix.

Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells.

The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells.

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H(2)O(2) generation.

Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal growth factor receptor in primary human keratinocytes. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H(2)O(2). Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal growth factor receptor phosphorylation and H(2)O(2) generation. Third, the structurally different antioxidants n-propyl gallate and N-acetylcysteine inhibited epidermal growth factor receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal growth factor receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal growth factor receptor phosphorylation and activation of extracellular-regulated kinase 1/2. These findings establish the following sequence of events: (1) H(2)O(2) generation, (2) epidermal growth factor receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H(2)O(2) as an important upstream mediator required for ligand-independent epidermal growth factor receptor phosphorylation and downstream signaling.

Effects of eight antibacterial agents on cell survival and expression of epithelial-cell- or cell-adhesion-related genes in human gingival epithelial cells.

Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes. These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.