Interleukin-1 (IL-1), originally defined as a product of activated macrophages, has since been found to be produced by many cell types including keratinocytes. The nature of this IL-1 activity in keratinocytes, originally known as epidermal cell-derived thymocyte activating factor (ETAF) has been the subject of many studies. In the course of this work it was found that the human keratinocyte cell line COLO 16 contains mRNA homologous to human monocyte-derived IL-1B. A 1.2 kbp cDNA was selected with a human IL-B probe from a lgt11 library constructed from COLO 16 mRNA. Sequence analysis revealed that this eDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1B. In addition, a partial cDNA (F8) was isolated from COLO 16 cells which has a distinct sequence from either IL-1a or B. There is evidence to suggest that the F8 message may be derived from differential splicing of a region of the human genome which also gives rise to the cGMP-gated ion channel in rod photoreceptor cells. The F8 cDNA hybridized on Northern blots of COLO 16 mRNA to a 1.6 kb message of low abundance. Antisera generated against a synthetic peptide based on inferred protein sequence from the cDNA reacted with a 20 and 30 kDa species in both COLO 16 cells and PMA-stimulated normal human keratinocytes. Expression of the partial cDNA in COS-1 cells resulted in activity in the thymocyte co-stimulation and D10.G4.1 T-cell stimulation assays, suggesting that ETAF activity may be due to a combination of IL-1 and F8. / Thesis / Master of Science (MS)

No / The ability of a cell to contract plays an important role in determining the ability of the cell to migrate, proliferate and associating with other cells. The transduction of the force in soft substrate such as the liquid crystal surface is a method proposed to study the traction forces of single cells. In this work, finite element method was used to study the compressive forces induced by the keratinocyte to the liquid crystal surface via the anchorage of focal contacts. The constitutive finite element model of the liquid crystal-focal contacts was established. The stress and displacement were analyzed using linear static stress analysis for a quiescent cell. The data for lateral displacements obtained from the experiment were provided as inputs to develop the model and verified through the output acquired for both simulation and experiment. The simulation results indicated that the cell compressive stresses were in the range of 14.93 ± 1.9 nN/μm2 per focal contact. Based on the result obtained, it was suggested to model focal contact-liquid crystal interface with a compressive model that can better approximate the mechanism observed

La phase de l'épithélialisation de la réparation cutanée se déroule en impliquant plusieurs processus dynamiques et interactifs pendant lesquels les kératinocytes migrent, prolifèrent et se différentient afin de reconstruire la fonction de la barrière. La migration des kératinocytes est l'événement qui détermine l'efficacité du processus entier. Le comportement migratoire est contrôlé au même temps au niveau extracellulaire et intracellulaire et dépend d'interactions dynamiques entre les cellules et leur environnement extracellulaire, des facteurs de croissance et des cytokines. Parmi les protéines de la matrice extracellulaire, la laminine 332 est un substrat d'adhésion majeur des kératinocytes qui joue un rôle important au cours de la migration des kératinocytes, travers son domaine LG4/5 localisé à l'extrémité carboxy-terminale de sa chaine a. Des études récentes ont rapporté que l'induction de la migration des kératinocytes par LG4/5 est dépendante des Métalloprotéinases Matricielles pro-migratoires (MMP)-9 et -1 qui jouent des rôles essentiels au cours de la cicatrisation et surtout pendant la ré-épithélialisation. Etant donné que des travaux antérieurs du laboratoire ont montré que le domaine LG4/5 participe à la dynamique du cytosquelette et à la motilité cellulaire au travers de liaisons avec les récepteurs de type de protéoglycanes à heparane sulfate, syndécan-1 et -4 on a regardé l'implication potentielle de ces récepteurs au processus. Afin d'analyser la participation possible des syndecans dans ce processus, nous avons développé une approche de mutagénèse dirigée dans la protéine LG4/5 recombinante pour altérer les sites de liaison aux syndécan-1 ou -4. Notre analyse PCR et nos résultats de zymographie ont révélé une différence du profile d'activation des MMPs en fonction de la mutation produite et donc de la capacité de la protéine à recruter le syndécan-1 ou le syndécan-4, ainsi que le syndécan-1, et pas la syndécan-4, est impliqué dans l'activation de la production de la MMP-9 par LG4/5. Nous avons ensuite confirmé ces résultats en réduisant l'expression du syndécan-1 dans des kératinocytes et on a pu aussi montrer que le traitement avec des cytokines telles que TNFalpha et IL-1beta, connues pour leur capacité d'induire l'activation de la MMP-9, a produit le même résultat dans ce systéme. L'addition de l'héparine dans nos experiences a inhibé l'activation de l'expression de MMP-9 suggerant que les heparanes sulfates dans syndecan-1 sont impliqué au mécanisme. Pour confirmer ces résultats des experiences avec des séries de syndecan-1 mutés sont en cours. Pour conclure, nos résultats montrent pour la première fois un rôle important de syndecan-1 à l'expression de MMP-9 suggérant que sa re-distribution au front des kératinocytes migratoires puisse éventuellement être liée au clivage ou à la dégradation des protéines de la matrice extracellulaire. En plus, nos résultats proposent que le domain LG4/5 de la laminin 332 libéré soit capable d'affecter la balance de l'expression de la MMP-9 lors de la migration des kératinocytes en leur permettant de traverser le caillot de fibrine / During skin repair, the epithelialization phase occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the barrier function. Keratinocyte migration determines the efficiency of the overall wound repair process. The migratory behaviour is governed at both the extracellular and intracellular levels and depends on the carefully balanced dynamic interactions of the cells with ECM components, growth factors and cytokines. Among extracellular matrix proteins, laminin 332, known as a major adhesion substrate for keratinocytes was shown to contribute to skin reepithelialization through its a3 chain C-terminal domains LG45. Recent studies have reported that LG45 induces keratinocyte migration, an event that relies on the involvement of the pro-migratory matrix metalloproteinases-1 and -9, two MMPs known to play a role in the reepithelialization phase of wound healing. As findings from our laboratory have reported that LG45 domains participate in cytoskeleton dynamic and cell movement through binding of the heparan sulphate proteoglycans syndecan-1 and -4, we analyzed the potential involvement of these receptors in this process. To that end, we have developed a site-directed mutagenesis approach within a recombinant LG45 protein to alter either the syndecan-1 or syndecan-4 binding site. Our PCR analysis and zymography results revealed that depending on the mutants, syndecan-1 or syndecan-4 recruitment induced different MMP activation profile and suggested that syndecan-1 plays a role in LG45 induced MMP-9 expression and activation. We confirmed these results by down regulating syndecans expression in keratinocytes and revealed that this phenomenon also occurred when cells were treated with TNFalpha or IL1beta, two cytokines known to up-regulate MMP-9 expression. Addition of heparin in these experiments abolished MMP-9 expression activation suggesting that syndecan-1 heparan sulfate moieties are involved in this mechanism. Confirming experiments using a series of mutated syndecan-1 in their ectodomain (lacking glycosaminoglycan chains) or in their cytoplasmic tail are ongoing in the lab. Taken together, our data demonstrate for the first time that syndecan-1 plays a pivotal role in MMP-9 expression, suggesting that its re-distribution at the front edge of migrating keratinocyte may have a role to play in the cleavage or degradation of extracellular matrix proteins. Our results further suggest that the released laminin 332 LG45 domain has the ability to impact the MMP9 expression balance during keratinocyte migration therefore facilitating their path through the fibrin clot

Skin wound healing presents a challenging and expensive clinical problem with nearly 20 million wounds requiring intervention leading to an annual cost of more than $8 million. Tissue engineered skin substitutes are valuable not only as a clinical therapy for chronic wounds and severe traumas, but also as in vitro 3D model systems to investigate wound healing and skin pathogenesis. However, these substitutes are limited by a lack of topography at the dermal-epidermal junction (DEJ). In contrast, the native DEJ is characterized by a series of dermal papillae which project upward into the epidermal layer and create physical topographic microniches that support keratinocyte stem cell clustering. In this thesis, we created novel 3D skin model systems to investigate the role of microtopography in regulating keratinocyte function and cell fate using scaffolds containing precisely engineered topographic features. We hypothesized that the microtopography of the DEJ creates distinct keratinocyte microniches that promote epidermal morphogenesis and modulate keratinocyte stem cell clustering which can be harnessed to create a more robust skin substitute that expedites wound closure. Using photolithographic techniques, we created micropatterned DEJ analogs and micropatterned dermal-epidermal regeneration matrices (µDERM) which couple a dermal support matrix to a micropatterned DEJ analog. We found that the incorporation of microtopography into our in vitro skin model resulted in a thicker, more robust epidermal layer. Additionally, we identified three distinct functional keratinocyte niches: the proliferative niche in narrow channels, the synthetic niche in wide channels and the keratinocyte stem cell niche in narrow channels and corner topographies. Ultimately, incorporation of both narrow and wide channels on a single construct allowed us to recreate native keratinocyte stem cell patterning in vitro. These model systems will allow us to investigate the role of cellular microniches in regulating cellular function and epidermal disease pathogenesis as well as to identify topographic cues that enhance the rate of wound healing.

As human epithelial cells age in culture, protein levels of the tumor suppressor protein p16INK4a continue to increase resulting in growth arrest independent of telomere length. Telomere-independent senescence induced by the p16INK4a/Rb tumor suppressor pathway prevents many epithelial cells from becoming immortalized by telomerase alone. Differences in culture conditions have been hypothesized to modulate both p16INK4a expression and replicative capacity of human epithelial cells; however, the mechanism(s) of p16INK4a regulation under these conditions is unknown. We have demonstrated that p16INK4a expression is delayed and replicative capacity increased in human keratinocytes grown in co-culture with post-mitotic, fibroblast feeder cells as compared to keratinocytes grown on tissue culture plastic alone. We have found that p16INK4a induction in human keratinocytes cultured on plastic alone is associated with a migratory phenotype and that p16INK4a expression is selectively induced in cells possessing markers of keratinocyte migration. Furthermore, we have identified that tyrosine kinase activity and proper functioning of the urokinase plasminogen activation system are required for p16INK4a induction during keratinocyte migration whereas specific signaling through either Src-PTKs or FAK does not appear to regulate this phenomenon. We have shown that human keratinocytes possessing telomerase activity and co-cultured with feeder cells do become immortal without any apparent cellular crisis. In contrast to previous reports, however, we demonstrate that telomerase immortalized keratinocytes co-cultured with feeders do not consistently growth arrest upon transfer to the plastic culture condition and display an increased frequency of p16INK4a promoter methylation. In summary, p16INK4a-induced, telomere-independent senescence is associated with an epithelial migration response and provides a significant proliferation barrier to epithelial cell immortalization regardless of culture conditions. These results provide new insights into p16INK4a regulation and have significant implications for the study of epithelial tumor cell invasion and telomerase reactivation therapies.

No description available.

The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient. The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjunction with growth factors and growth factor-binding proteins (VN:GF combination), to promote enhanced cell migration and growth through the co-activation of integrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types. Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation. Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.

2-Chloroethyl ethyl sulfide (CEES) or half-mustard gas, a sulfur mustard (HD) analog, is a genotoxic agent that causes oxidative stress and induces both apoptotic and necrotic cell death. Sodium pyruvate induced a necrosis-to-apoptosis shift in HaCaT cells exposed to CEES levels ≥ 1.5 mmol/L and lowered markers of DNA damage, oxidative stress, and inflammation. This study provides a rationale for the future development of multicomponent therapies for HD toxicity in the skin. We hypothesize that a combination of pyruvates with scavengers/antioxidants encapsulated in liposomes for optimal local delivery should be therapeutically beneficial against HD-induced skin injury. However, the latter suggestion should be verified in animal models exposed to HD.

Indiana University-Purdue University Indianapolis (IUPUI) / Human skin, consisting of the outer epidermis and inner dermis, serves as a barrier that protects the body from an onslaught of environmental stresses. Keratinocytes in the stratified epidermis undergo sequential differentiation that consists of multiple layers of cells differing in structure and function. Therefore, keratinocytes must not only combat environmental stress, but need to undergo massive changes in gene expression and morphology to form a proper barrier. One mode by which cells cope with stress and differentiation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P), which causes global inhibition of protein synthesis coincident with preferential translation of select gene transcripts. Translational repression allows stressed cells to conserve energy and prioritize pro-survival processes to alleviate stress damage. Since eIF2α kinases are each activated by distinct types of stress, this pathway is referred to as the Integrated Stress Response (ISR). We sought to identify the roles of the ISR in the keratinocyte response to the stresses associated with differentiation and ultraviolet B (UVB) irradiation. In this thesis, we show that both general and gene-specific translational control in the ISR are activated following differentiation or UVB irradiation of human keratinocytes. ISR deficiency through genetic modifications or pharmacological interventions caused severe divergence from the appropriate keratinocyte response to differentiation or UVB. Differentiation genes were selectively translated by eIF2α-P, and inhibition of the ISR diminished their induction during differentiation. Furthermore, loss of the eIF2α kinase GCN2 (EIF2AK4) adversely affected the ability of keratinocytes to stratify in three dimensional cultures. Our analysis also revealed a non-canonical ISR response following UVB irradiation, in which downstream factors ATF4 (CREB2) and CHOP (DDIT3/GADD153) were poorly expressed due to repressed transcription, despite preferential translation in response to eIF2α-P. The ISR was cytoprotective during UVB and we found that eIF2α-P was required for a UVB induced G1 arrest, cell fate determination, and DNA repair via a mechanism involving translational control of human CDKN1A (p21 protein) transcript variant 4 mRNA. Collectively, this thesis describes novel roles for the ISR in keratinocyte differentiation and response to UVB, emphasizing the utility of targeting translational control in skin disease therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21634号 / 医博第4440号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM