Expression of keratinocyte biomarkers is governed by environmental biomechanics
Introduction
Cells of solid body tissues are capable of feeling and responding to their extracellular environment (Discher et al., 2005). This perception of environmental biomechanics, similar to growth factors induces intracellular signaling cascades, which lead to behavioral cellular responses. These responses, critical in tissue homeostasis and disease, include features such as the development of regular cell morphology, proliferation and differentiation (Ingber, 2003, Stark et al., 2006).
Response to the extracellular environment is determined by the stiffness of the extracellular matrix (ECM), reflected by the elasticity or Young's modulus (D'Sa et al., 2009). This modulus depends on the type of tissue, i.e. whether the matrix constituents harbor cells of soft, e.g. fibroblasts of soft connective tissues, or hard tissues, e.g. mineralized tissues, like cartilage or bone (Discher et al., 2005). Feeling of the biofunctional extracellular environment is based on adhesion of focal contact integrins to ECM molecules, which allocate distinct patterns of nano- and microscaled cell adhesion or anchor points (Mussig et al., 2010, Nicolas and Safran, 2006). By synergism, the ECM's Young's modulus and patterning of cell adhesion points substantiate cornerstones of environmental biomechanics.
In our body tissues, homeostasis is characterized by the controlled balance of cell proliferation and differentiation, whereat in solid tissues, like the corneal epithelium, ECM-driven anchorage-dependent regular cell morphogenesis is mandatory (Li et al., 1999). In epithelia, division or proliferation of stem cells and transient amplifying cells substitutes such cells, which were lost due to terminal differentiation, thereby guaranteeing lifelong maintenance of tissue renewal (Slack, 2000). Similar to skin or the oral cavity epithelia, the corneal epithelium comprises of several cell layers, thereby belonging to the complex stratified epithelia. Unlike skin or the oral cavity, in corneal epithelial differentiation the spatial trajectory of progressively differentiating cells is not reflected by an exclusive bottom-up migration, but rather an out-inside modus, i.e. centripetal from the limbal margins to the cornea center. Consequently, the proliferative compartment of the corneal epithelium localizes preferentially at the limbal margins and in the basal cell layer, but not in parabasal cell layers, like in oral epithelia (Papini et al., 2005). Despite these differences, progressive stages of corneal epithelial cell differentiation are characterized by the expression of tissue-specific biomarkers, among which members of the intermediate filament polypeptide family, the cytokeratins (KRT), indicate early and late stages of corneal keratinocyte differentiation. While limbus-associated KRT19 identifies early corneal keratinocyte differentiation (Espana et al., 2004, Schlotzer-Schrehardt and Kruse, 2005), progressive, i.e. later stages are indicated by KRT12 expression (Espana et al., 2004, Li et al., 2008, Liu et al., 1993). Biomarkers of terminal corneal differentiation are involucrin and filaggrin (Adhikary et al., 2004, Tong et al., 2006). As known from skin, involucrin, covalently cross-linked at inner plasma membrane sites, renders a protein scaffold, which in interplay with additional precursor proteins yields in the formation of the cornified envelope (Kalinin et al., 2002). In this envelope, the covalently cross-linked proteins provide reinforcement against external insults (Steinert and Marekov, 1995). Enzymatically, cornified envelope formation is managed by transglutaminase type I activity, with involucrin being a highly efficient enzyme substrate (Lambert et al., 2000).
During cornified envelope formation, transglutaminases are also involved in the cross-linkage of the condensed keratin cytoskeleton upon keratinocyte terminal differentiation. Cytoskeletal condensation is mediated by filaggrin (filament aggregating protein), which rapidly aggregates to the keratin cytoskeleton (Smith et al., 2006). As in other self-renewing tissues, also ocular surface epithelia, including cornea, are suggested to contain “side population” (SP) cells, which display features seen in multiple adult stem cell types. Among these features, efflux of the Hoechst fluorescence dye indicates SP cells, while the efflux driver, the G2 subtype member of the ATP Binding Cassette (ABC) transporter ABCG2, serves as a putative stem cell marker (Budak et al., 2005, Zhou et al., 2001).
In line with keratinocytes of other complex human epithelia, also cells derived from human corneal epithelium can hardly be propagated in long-term in vitro cell cultures, due to the cells’ innate limited life span. This problem can be abandoned by immortalization of primary human keratinocytes with viral DNA or distinct viral oncogenes, respectively. As shown by our own group for, one-time infection of human keratinocytes with the E6/E7 oncogenes of the human papilloma virus type 16 (HPV 16) has been proven an established principle for successful and smooth immortalization (Pear et al., 1993), i.e. gain of non-limited in vitro propagation in conjunction with preservation of the cell- and tissue-specific differentiation program (Roesch-Ely et al., 2006).
In this study, we show for the first time that defined environmental biomechanics governs features of cell behavior in immortalized human corneal keratinocytes (IHCK). By comprising large micropatterns of 11 μm, biomechanics revealed favoritism in expression of late and terminal keratinocyte differentiation biomarkers, concomitant with reduced proliferation propensity, increased levels of stem cell marker ABCG2 and less proper morphogenesis. The results suggest that these biomechanical conditions reflect a more inconvenient stressful extracellular microenvironment for IHCK.
Section snippets
Cell culture and immortalization
After informed consent and approval by the institutional ethics committee of the Medical Faculty, University of Freiburg (Vote number 307/09), primary human corneal keratinocytes were grown as explant culture from the limbus, which remained after keratoplasty procedures and was obtained from the Cornea Eye Bank of Freiburg. Small tissue fragments were grown as explant cultures with the epithelial side downward directed using DME medium (PAA, Cölbe, Germany) supplemented with 10% fetal calf
Primary human corneal keratinocytes immortalized with HPV 16 E6/E7 (IHCK) oncogenes exhibit preservation of tissue-specific biomarker expression
By analysis of tissue-specific biomarkers, we aimed at proving the suitability of IHCK as a corneal epithelial cell system. Therefore, we compared the expression of corneal markers, cytokeratin 19 (KRT19), cytokeratin 12 (KRT12), involucrin and filaggrin, in conjunction with ABCG2, β1 integrin and tri-methyl histone H3 by indirect immunofluorescence (IIF) on frozen sections of native cornea, with matched IHCK. As shown in Fig. 1, Fig. 2, the comparison revealed a coincidence in the expression
Discussion
In contrast to rodent epithelial cells, including cornea (Castro-Munozledo, 1994), spontaneous immortalization of human keratinocytes is rather unusual. In order to achieve successful immortalization, oncogenes such as the large T antigen of the simian virus type 40 and the E6 and E7 genes of the human papilloma virus type 16 (HPV16-E6/E7) have provided valuable tools for establishing long-term human keratinocyte cell cultures (Bryan and Reddel, 1994, Furukawa et al., 1996). To escape from
Acknowledgements
We are grateful to Prof. Dr. Holger Reinecke head of the laboratory of Process Technology of the Department of Microsystem Engineering University Freiburg for manufacturing the silicon wafers, necessary for PDMS pillar surface generation. This work was supported in part by grants from the Medical Faculty of the University of Freiburg to Philipp Eberwein and Pascal Tomakidi (FOKO: TOM 668/08).
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