A Limited Role for Retinoic Acid and Retinoic Acid Receptors RARα and RARβ in Regulating Keratin 19 Expression and Keratinization in Oral and Epidermal Keratinocytes

Different types of stratified squamous epithelia—for example, the “orthokeratinized” epidermis, the “parakeratinized” gingiva, and the “nonkeratinized” oral lining mucosal epithelia—are formed by intrinsically distinct keratinocyte subtypes. These subtypes exhibit characteristic patterns of keratin protein expression in vivo and in culture. Keratin 19 is an informative subtype-specific marker because the basal cells of only nonkeratinizing epithelia express K19 in vivo and in culture. Epidermal keratinocytes normally do not express K19, but can be induced to do so in culture by retinoic acid (RA). Keratinocyte subtypes express the retinoic acid receptor (RAR)β at levels roughly correlated with their level of K19 expression in culture and their potential for forming a nonkeratinized epithelium in vivo. We tested the hypothesis that the level of RARβ expressed by a keratinocyte determines its K19 expression and its form of suprabasal differentiation. Normal human epidermal and gingival keratinocytes stably overexpressing either RARβ or RARα were generated by defective retroviral transduction. Overexpression of either receptor enhanced the RA inducibility of K19 in conventional culture, in that the proportion of the transductants becoming K19+ in response to RA was markedly increased compared with controls. The pattern of differentiation of the epithelium formed in organotypic culture, assessed by basal K19 and suprabasal K1, K4, and filaggrin expression, however, was unaltered by PAR overexpression. Thus, the susceptibility of keratinocytes to regulation of K19 expression by retinoids is conditional, and levels of neither RARβ nor RARα are limiting to the intrinsic mechanism that specifies alternate differentiation pathways for stratified squamous epithelia

RSK Activation of Translation Factor eIF4B Drives Abnormal Increases of Laminin γ2 and MYC Protein during Neoplastic Progression to Squamous Cell Carcinoma

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5′ untranslated regions (5′-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5′-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC

Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction

Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation

Background: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum

Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs

Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers

Rivkah Isseroff and colleagues describe how stress-induced elevation of epinephrine levels can impair the healing of burns in mice and suggest that β2 adrenergic receptor antagonists may have a role in improving skin wound repair

Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable--keratinocyte-derived--cytokine and growth factor milieu. In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß. Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis

INTRODUCTION: There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. METHODS: We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. RESULTS: Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. CONCLUSIONS: These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis