No description available.

Atopic eczema is a T-lymphocyte mediated chronic inflammatory skin disorder. The interaction of CD4+ T-lymphocytes with epidermal keratinocytes results in dysregulated, chronic inflammation and altered barrier function. T-lymphocyte induced keratinocyte apoptosis has been proposed as a mechanism by which epidermal integrity is impaired in eczema. Apoptosis of keratinocytes is thought to result from Tlymphocyte associated Fas ligand (FasL) binding to the death receptor Fas on keratinocytes. The primary aim of this project was to characterize the induction of keratinocyte apoptosis by T-lymphocytes and address the hypothesis that insulin-like growth factor-I (IGF-1), transforming growth factor [beta]1 (TGF[beta]1) and a milk derived growth factor extract containing TGF[beta] and IGF-I (whey growth factor extract; WGFE) protect keratinocytes from T-lymphocyte mediated apoptosis. To address the aims of this project, an in vitro co-culture model was developed combining T-lymphocytes with keratinocytes. Co-cultures were initially established using human Jurkat T-lymphocytes and human HaCaT keratinocytes with more extensive characterisation undertaken using primary CD4+ T-lymphocytes together with HaCaTs or normal human epidermal keratinocytes (NHEK). Annexin V and propidium iodide staining was established as the primary method for measuring keratinocyte apoptosis with this validated using sodium butyrate a known inducer of apoptosis. Changes in nuclear fragmentation and cell morphology were also examined as a key feature of apoptosis. The involvement of the Fas pathway was investigated by assessing T-lymphocyte FasL expression, keratinocyte Fas expression and downstream caspase activation. Inflammatory cytokines IFN[gamma] and TNF[alpha] were also examined due to their ability to induce Fas expression. Studies performed with T-lymphocytes demonstrated that keratinocyte apoptosis was induced, with this due primarily to direct T-lymphocytes and keratinocytes interactions, rather than soluble mediators in the co-culture milieu. Activated T-lymphocytes were found to have high levels of FasL and to upregulate keratinocyte Fas expression. The increased keratinocyte Fas was associated with increased IFN[gamma] levels in the co-culture media and activation of the caspase cascade. A Fas blocking antibody prevented T-lymphocyte induced keratinocyte apoptosis demonstrating that this was a Fas dependent event. As the primary function of keratinocytes is to terminally differentiate, the differentiation status of the cells induced to undergo apoptosis was examined. It was demonstrated that T-lymphocytes decrease the intensity of ?6 integrin expression by the keratinocytes. This marker identifies undifferentiated basal cells as high expressors of [alpha]6, with cells in the early stages of differentiation pathway found to be low expressors of [alpha]6. Co-staining with Annexin V demonstrated that the apoptotic keratinocytes were low expressors of [alpha]6 and thus cells committed to the early stages of differentiation. This suggested that the T-lymphocytes initiated the onset of keratinocyte terminal differentiation with this linked to the cells being more susceptible to death induced by T-lymphocyte by activation of the Fas pathway. The ability of TGF[beta]1, IGF-I and WGFE to inhibit T-lymphocyte induced keratinocyte apoptosis was examined. A combination of recombinant TGF[beta] (10ng) & IGF-I (100ng) was able to significantly inhibit keratinocyte apoptosis. A similar result was obtained with WGFE, and although these growth factor treatments were able to reduce the elevated IFN[gamma] levels in the co-culture media, they did not reduce T-lymphocyte induced Fas upregulation. The TGF?1 and IGF-I combination as well as WGFE did however prevent the T-lymphocyte induced shift from [alpha]6 bright to dim expressing keratinocytes. As such, the growth factor combinations appeared to protect the keratinocytes from T-lymphocyte mediated apoptosis by preventing them from committing to terminal differentiation. The studies in this thesis have characterised the Fas associated mechanisms by which T-lymphocytes induce keratinocyte apoptosis and suggest specific growth factor combinations may have the potential to ameliorate the reduced barrier function associated with inflammatory skin conditions such as atopic eczema.

Objectives: Periodontal disease is a chronic inflammation resulting in periodontal attachment loss. Keratinocyte Growth Factor-1 (KGF-1) is upregulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific Keratinocyte Growth Factor Receptor (KGFR). First, we examined KGF-1 and KGFR expression and localization in human periodontal tissues. Second, we extended these studies by developing an in vitro mechanical wound model to mimic trauma to the periodontal pocket epithelium and examined ligand independent KGFR activation and cell migration. Methods: In our study of human gingival tissues, we used immunohistochemistry and laser capture microdissection with RT-PCR to analyze KGF-1 and KGFR expression and localization. To study ligand independent KGFR phosphorylation, KGFR internalization along the wound edge was imaged using immunohistochemical staining and KGFR phosphorylation confirmed using immunoprecipitation with western blotting. Wounding induced oxidative stress was detected using DCFH-DA (2',7'-dichlorofluorescin diacetate) and modulated by pretreatment with an antioxidant. Changes in migration were examined in the presence or absence of pathway specific inhibitors. Results: KGF-1 protein localized to areas of junctional and basal oral epithelial cells was significantly increased in periodontal pocket epithelium (p<0.01) and oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, and was increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmedKGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. In our cell culture model, mechanical wounding induced ligand independent KGFR activation. ROS (Reactive Oxygen Species) generation along the wound edge was associated with KGFR activation and scavenging of ROS reduced KGFR phosphorylation. The c-Src family inhibitor, PP1, significantly inhibited KGFR phosphorylation. Functionally cell migration was reduced by PP1 (82.7%), SU5402(70%) and PD98059 (57%). Conclusions: KGF-1 and KGFR proteins are expressed in health but significantly induced in human diseased periodontal tissues. Microwounding associated generation of ROS mediates KGFR phosphorylation via c-Src kinase signaling and induced wound edge cell migration. Therefore, regulation of epithelial cell behavior associated with the onset and progression of periodontal disease may possibly be mediated by two related but distinct mechanisms. (1) Ligand-dependent activation of KGFR due to upregulation of KGF-1. (2) Ligand-independent activation of KGFR due to chronic microwounding.

Tumor Susceptibility Gene 101, TSG101, exhibits multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the signals involve in the regulation of TSG101 gene functions are unclear. In this present study, we observed congruous TSG101 up-regulation and the differentiation status of keratinocyte in both human foreskin tissue and reconstructed organotypic skin culture. In addition, we found an essential and downstream role of TSG101 in calcium-induced early keratinocyte differentiation since TSG101 siRNA inhibits this process. Our results also indicate a PKC-dependent mechanism is involved based on the following findings. First, a PKC agonist, TPA up-regulates TSG101 and keratin 10 under low calcium condition. Second, co-treatment of keratinocytes with GF 109203X, a PKC inhibitor, blocks TPA-induced TSG101 and keratin 10 up-regulation. Previous report indicates TSG101 gene exhibits a TATA-less and Sp1-containing promoter. Our analysis further shows that both calcium and TPA stimulate phosphorylation of Sp1 and the corresponding TSG101 wild type promoter activity, but not the activity of Sp1 site mutant TSG101 promoter. The co-treatment with GF 109203X blocks the above effects of calcium and TPA, implying that this is a PKC signaling-dependent process. Taken together, these data suggest a PKC-Sp1 signaling is involved in early differentiation switch of keratinocyte through up-regulation of TSG101. Functional inactivation experiment indicates that tsg101 is a tumor suppressor in mouse model. However, many studies using human tumor specimens or conditional knockout mouse give discrepant and contradictive results. Therefore, the role of TSG101 in human cancer remains illusive. Here we demonstrate an inverse correlation between TSG101 and p16INK4a or acetylated- histone H4 protein expression profiles in human head and neck squamous cell carcinomas (HNSCC) (N=98, p<0.001). Using conditioned human HEp2 cells, we confirm that TSG101 negatively modulates p16INK4a expression. Chromatin immunoprecipitation and the subsequent PCR analysis reveal that TSG101 dose-dependently decreases the amount of acetylated histone H4-associated chromatin on p16INK4a promoter. In addition, TSG101 interacts and colocalizes with HDAC1 and SUMO-1 in the nucleus. Furthermore, TSG101 confers a dose-dependent effect on promoting HDAC1 SUMOylation, hence its activity. Taken together, our data demonstrate for the first time that TSG101 can promote SUMO-1 modification of HDAC1, which impacts on down-regulation of p16INK4a gene expression, providing evidence whereby TSG101 might participate in the epigenetic silencing of p16INK4a during the development of HNSCC.

Objectives: Periodontal disease is a chronic inflammation resulting in periodontal attachment loss. Keratinocyte Growth Factor-1 (KGF-1) is upregulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific Keratinocyte Growth Factor Receptor (KGFR). First, we examined KGF-1 and KGFR expression and localization in human periodontal tissues. Second, we extended these studies by developing an in vitro mechanical wound model to mimic trauma to the periodontal pocket epithelium and examined ligand independent KGFR activation and cell migration. Methods: In our study of human gingival tissues, we used immunohistochemistry and laser capture microdissection with RT-PCR to analyze KGF-1 and KGFR expression and localization. To study ligand independent KGFR phosphorylation, KGFR internalization along the wound edge was imaged using immunohistochemical staining and KGFR phosphorylation confirmed using immunoprecipitation with western blotting. Wounding induced oxidative stress was detected using DCFH-DA (2',7'-dichlorofluorescin diacetate) and modulated by pretreatment with an antioxidant. Changes in migration were examined in the presence or absence of pathway specific inhibitors. Results: KGF-1 protein localized to areas of junctional and basal oral epithelial cells was significantly increased in periodontal pocket epithelium (p<0.01) and oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, and was increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmedKGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. In our cell culture model, mechanical wounding induced ligand independent KGFR activation. ROS (Reactive Oxygen Species) generation along the wound edge was associated with KGFR activation and scavenging of ROS reduced KGFR phosphorylation. The c-Src family inhibitor, PP1, significantly inhibited KGFR phosphorylation. Functionally cell migration was reduced by PP1 (82.7%), SU5402(70%) and PD98059 (57%). Conclusions: KGF-1 and KGFR proteins are expressed in health but significantly induced in human diseased periodontal tissues. Microwounding associated generation of ROS mediates KGFR phosphorylation via c-Src kinase signaling and induced wound edge cell migration. Therefore, regulation of epithelial cell behavior associated with the onset and progression of periodontal disease may possibly be mediated by two related but distinct mechanisms. (1) Ligand-dependent activation of KGFR due to upregulation of KGF-1. (2) Ligand-independent activation of KGFR due to chronic microwounding.

Objectives: Periodontal disease is a chronic inflammation resulting in periodontal attachment loss. Keratinocyte Growth Factor-1 (KGF-1) is upregulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific Keratinocyte Growth Factor Receptor (KGFR). First, we examined KGF-1 and KGFR expression and localization in human periodontal tissues. Second, we extended these studies by developing an in vitro mechanical wound model to mimic trauma to the periodontal pocket epithelium and examined ligand independent KGFR activation and cell migration. Methods: In our study of human gingival tissues, we used immunohistochemistry and laser capture microdissection with RT-PCR to analyze KGF-1 and KGFR expression and localization. To study ligand independent KGFR phosphorylation, KGFR internalization along the wound edge was imaged using immunohistochemical staining and KGFR phosphorylation confirmed using immunoprecipitation with western blotting. Wounding induced oxidative stress was detected using DCFH-DA (2',7'-dichlorofluorescin diacetate) and modulated by pretreatment with an antioxidant. Changes in migration were examined in the presence or absence of pathway specific inhibitors. Results: KGF-1 protein localized to areas of junctional and basal oral epithelial cells was significantly increased in periodontal pocket epithelium (p<0.01) and oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, and was increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmedKGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. In our cell culture model, mechanical wounding induced ligand independent KGFR activation. ROS (Reactive Oxygen Species) generation along the wound edge was associated with KGFR activation and scavenging of ROS reduced KGFR phosphorylation. The c-Src family inhibitor, PP1, significantly inhibited KGFR phosphorylation. Functionally cell migration was reduced by PP1 (82.7%), SU5402(70%) and PD98059 (57%). Conclusions: KGF-1 and KGFR proteins are expressed in health but significantly induced in human diseased periodontal tissues. Microwounding associated generation of ROS mediates KGFR phosphorylation via c-Src kinase signaling and induced wound edge cell migration. Therefore, regulation of epithelial cell behavior associated with the onset and progression of periodontal disease may possibly be mediated by two related but distinct mechanisms. (1) Ligand-dependent activation of KGFR due to upregulation of KGF-1. (2) Ligand-independent activation of KGFR due to chronic microwounding. / Dentistry, Faculty of / Graduate

No description available.

Wounding of the cornea generates lateral electric fields (EFs) and initiates the expression of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF; Chiang et al., 1992; Wilson et al., 1999a). Therefore, these biologically generated EFs and endogenous growth factors may be of particular significance during wound healing. In the presence of an EF (150 mV/mm) cultured corneal epithelial cells (CECs) oriented perpendicular, directed cathodally and migrated at an enhanced rate. The induction times, induction thresholds and response patterns for these behaviours in increasing field strengths, indicated that they may operate through separate and parallel pathways. The application of either HGF or KGF enhanced the rate of CEC migration but neither affected the extent of CEC orientation or directionality. The distribution of HGF receptors (HGFR) was found to be exclusive to the cell body in the presence of an EF, the receptors accumulated cathodally. Moreover, the asymmetrical accumulation of HGFR in the presence of an EF correlated with the direction of CEC migration. The application of both HGF and an EF activated extracellular-signal regulated kinase (ERK) a mitogen-activated protein kinase. Furthermore, in the presence of an EF the observed ERK activation was greater in the cathodal facing half of the CECs. Inhibition of ERK reduced the extent of HGF and EF-enhanced CEC migration rate but did not alter EF-induced CEC cathodal directionality or perpendicular orientation. The HGF- and EF-enhancement of CEC migration rate may involve the activation of ERK and with the downstream liberation of leukotrienes and phosphorylation of MLCK, would culminate in actin contraction and polymerisation respectively. The potential clinical relevance for this work would concern the topical application of HGF and exogenous application of EFs to corneal epithelial wounds in order to augment healing in patients where this process is slow or deficient.

The migration of keratinocytes across wound beds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease, and reactive oxygen species damage accumulation increases, but it is unclear if these factors relate specifically to migration of human skin keratinocytes (HSKs). In this study, two concentrations of oxygen (4% and 21%) were used to model low and high oxidative stress to produce varying levels of reactive oxygen species. When migration of HSKs from young and old primary skin were compared by scratch assay, those from old skin migrated faster in high oxygen tension than did young HSKs, which was an opposite trend from that seen in young skin. An intense increase in reactive oxygen species at margins immediately after scratching was seen in both young and old HSKs, but reactive oxygen species disappeared from young skin at 21% oxygen most quickly. These cells also had the slowest migration. These findings suggest that old and young keratinocytes respond differently to oxidative stress, and that migration of keratinocytes--a key step in re-epithelialization of wounds, is effected by the efficacy of reactive oxygen species removal.

No description available.