Browsing School, Graduate by Subject "Keratinocytes"
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Abutment Material Affects the Attachment of Co-cultured Fibroblasts and KeratinocytesCreating a stronger transmucosal seal around the implant abutment may help prevent epithelial downgrowth and resultant crestal bone loss. Most studies focus on titanium and zirconium, but provisional restorations are gaining popularity and these materials require further study. Previous in vitro models utilize a monoculture technique to understand cell behavior, which makes direct intercellular comparisons difficult. Our first aim was to develop a co-culture of human gingival fibroblasts and human oral keratinocytes. Then, cell attachment, proliferation, and migration across six commercially available abutment materials was ascertained and comparisons drawn. Discs made of smooth titanium, (control), rough titanium, CAD/CAM poly (methylmethacrylate), poly ether etherketone, smooth zirconium, and rough zirconium were chosen. Preliminary results indicate that at various time points, significant differences in fibroblast and keratinocyte proliferation and attachment exist among abutment materials.
Matriptase in Skin: Function and RegulationEpidermal differentiation is a carefully orchestrated process that leads to the formation of the critical protective barrier provided by the skin. The process of generating a functional epidermal layer requires progressive remodeling of cell morphology and tissue structure, and involves significant pericellular proteolysis that must be regulated in a precisely controlled manner. In particular, the matriptase-driven protease network plays a critical role in epidermal barrier construction as well as in the regenerative processes required for wound healing. In this dissertation, I have identified and characterized novel molecular mechanisms governing the regulation of matriptase, and cellular mechanisms by which matriptase activates its molecular targets and contributes to keratinocyte differentiation and formation of epidermal barrier. First, I identified plasminogen as a keratinocyte-selective extracellular stimulus for matriptase activation. The discovery of plasminogen as an initiating signal of the protease cascade reaffirms the theory that the matriptase-uPA-plasmin cascade is not unidirectional in the activation of its components, but it is reciprocal. In addition to HAI-1, I also revealed keratinocytes employ antithrombin as a significant endogenous protease inhibitor. The enhanced role of antithrombin in matriptase inhibition in keratinocytes reveals the regulatory adaptation in stratified epithelial cells due to the changes in tissue structure, compared to the polarized epithelial cells. With the dual inhibitory mechanisms, I further revealed that matriptase acts on its molecular targets in two different ways: a rapid activation of prostasin by cell-associated active matriptase under extremely tight control of HAI-1, and the action on several other substrates, including uPA, HGF, and syndecan-1, by secreted active matriptase that is controlled by antithrombin. Last but not least, I also demonstrated that the physiological role of matriptase in human skin likely lies in the basal and spinous keratinocytes that are involved in proliferation and early differentiation. The role of matriptase in the early stages of keratinocyte life history was further supported by the increased matriptase zymogen activation in the keratinocytes of the bulge area. By charactering the prominent regulators, downstream effectors, and the expression and activation states of matriptase in human skin, a clearer picture is emerging regarding the role of matriptase in skin biology.
Role of p38δ and MEP50 in Epidermal Keratinocyte HomeostasisKeratinocytes are the major cell type in the human epidermis, the outer layer of skin. These cells undergo a tightly regulated terminal differentiation program which results in the formation of the cornified envelope. This envelope is essentially the barrier which protects us from mechanical stress, extreme climate conditions and infectious agents. A fully functional epidermal barrier dictates a perfect balance between proliferation and differentiation. PKCδ, a novel PKC isoform and a key controller of epidermal differentiation regulates proliferation by increasing p21Cip1 expression. However, little is known about the mediators involved in this regulation. We hypothesize that p38δ MAPK, a downstream kinase is a mediator in this process. We observe that p38δ regulates p21Cip1 mRNA/protein levels in a p53 dependent manner. Additionally, PKCδ selectively activates p38δ and treatment with p38δ-siRNA or dominant negative p38 results in attenuation of the PKCδ induced p21Cip1 response. Furthermore, p53 is identified as a novel target of p38δ and is a key mediator in the p38δ -p21Cip1 signaling cascade. Moreover, the physiological relevance of this pathway is confirmed in the PKCδ and p38δ knockdown organotypic cultures which are thicker, have additional layers and reduced cornified envelope formation as compared to the controls. We have also identified the Protein Arginine Methyltransferase (PRMT5) and Methylosome Protein 50 (MEP50) as novel targets of PKCδ and p38δ. MEP50 enhances keratinocyte proliferation and opposes differentiation by mechanisms involving silencing p21Cip1 and involucrin. We found that symmetric demethylation of arginines in histones H3 and H4 plays a key role in this regulation. Additionally, the pro-proliferation role of MEP50 is physiologically relevant as MEP50 knockdown rafts are significantly thinner and have fewer number of Ki67 positive cells than the control rafts. These studies highlight p38δ as a common kinase regulating the dual processes of proliferation and differentiation and characterize the role of MEP50/PRMT5 as novel players in controlling epidermal homeostasis.