Molecular Genetics and Genomics

, Volume 284, Issue 1, pp 1–9 | Cite as

Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent

  • Joseph Mazar
  • Satyabrata Sinha
  • Marcel E. Dinger
  • John S. Mattick
  • Ranjan J. PereraEmail author
Original Paper


The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and cornified layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, differentiation, and cell death. To determine genes responsible for initiating and maintaining a cornified epidermis, organotypic cultures comprised entirely of stratified KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array profile of submerged cultures containing KCs predominantly in a proliferative (relatively undifferentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct profile of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, differentiation, and cell death. Next, differentially expressed microRNAs (miRNAs) and long non-coding (lncRNA) RNAs were identified in EEs. Several differentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but down-regulated by 120 h. To study the lncRNA regulation in EEs, we profiled lncRNA expression by microarray and validated the results by qRT-PCR. Although the differential expression of several lncRNAs is suggestive of a role in epidermal differentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin.


Keratinocytes Differentiation mRNA Transcriptome miRNA 



We would like to thank Laura Brovold and Ally Perlina from GeneGo, Inc. ( for assistance with systems level network mapping and the Affymetrix chip design team for making the custom miRNA array and protocols. We also would like to thank the Invitrogen/Life Technologies epigenetic gene regulation team for miRNA and lncRNA NCode array support and Dr. Brian Nickoloff for providing RNA for expression analysis and Fig. 1 for EE differentiation. Debbie McFadden provided assistance in formatting the manuscript.

Supplementary material

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Supplementary material 1 (TIFF 145 kb)
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Supplementary material 2 (TIFF 207 kb)
438_2010_543_MOESM3_ESM.tif (174 kb)
Supplementary material 3 (TIFF 173 kb)


  1. Amaral PP, Dinger ME, Mercer TR, Mattick JS (2008) The eukaryotic genome as an RNA machine. Science 319:1787–1789CrossRefPubMedGoogle Scholar
  2. Barker JN, Mitra RS, Griffiths CE, Dixit VM, Nickoloff BJ (1991) Keratinocytes as initiators of inflammation. Lancet 337:211–214CrossRefPubMedGoogle Scholar
  3. Boehm I (2006) Apoptosis in physiological and pathological skin: implications for therapy. Curr Mol Med 6:375–394CrossRefPubMedGoogle Scholar
  4. Burchiel SW, Thompson TA, Lauer FT, Oprea TI (2007) Activation of dioxin response element (DRE)-associated genes by benzo(a)pyrene 3, 6-quinone and benzo(a)pyrene 1, 6-quinone in MCF-10A human mammary epithelial cells. Toxicol Appl Pharmacol 221:203–214CrossRefPubMedGoogle Scholar
  5. Candi E, Schmidt R, Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6:328–340CrossRefPubMedGoogle Scholar
  6. Carninci P, Sandelin A, Lenhard B, Katayama S, Shimokawa K, Ponjavic J, Semple CA, Taylor MS, Engstrom PG, Frith MC, Forrest AR, Alkema WB, Tan SL, Plessy C, Kodzius R, Ravasi T, Kasukawa T, Fukuda S, Kanamori-Katayama M, Kitazume Y, Kawaji H, Kai C, Nakamura M, Konno H, Nakano K, Mottagui-Tabar S, Arner P, Chesi A, Gustincich S, Persichetti F, Suzuki H, Grimmond SM, Wells CA, Orlando V, Wahlestedt C, Liu ET, Harbers M, Kawai J, Bajic VB, Hume DA, Hayashizaki Y (2006) Genome-wide analysis of mammalian promoter architecture and evolution. Nat Genet 38:626–635CrossRefPubMedGoogle Scholar
  7. Chaturvedi V, Sitailo LA, Bodner B, Denning MF, Nickoloff BJ (2006) Defining the caspase-containing apoptotic machinery contributing to cornification in human epidermal equivalents. Exp Dermatol 15:14–22CrossRefPubMedGoogle Scholar
  8. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179CrossRefPubMedGoogle Scholar
  9. Dinger ME, Amaral PP, Mercer TR, Pang KC, Bruce SJ, Gardiner BB, Askarian-Amiri ME, Ru K, Solda G, Simons C, Sunkin SM, Crowe ML, Grimmond SM, Perkins AC, Mattick JS (2008) Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation. Genome Res 18:1433–1445CrossRefPubMedGoogle Scholar
  10. Eckert RL, Crish JF, Robinson NA (1997) The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation. Physiol Rev 77:397–424PubMedGoogle Scholar
  11. Garces C, Ruiz-Hidalgo MJ, Font de Mora J, Park C, Miele L, Goldstein J, Bonvini E, Porras A, Laborda J (1997) Notch-1 controls the expression of fatty acid-activated transcription factors and is required for adipogenesis. J Biol Chem 272:29729–29734CrossRefPubMedGoogle Scholar
  12. Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458:223–227CrossRefPubMedGoogle Scholar
  13. Harris IR, Siefken W, Beck-Oldach K, Wittern KP, Pollet D (2002) NAD(P)H:quinone reductase activity in human epidermal keratinocytes and reconstructed epidermal models. Skin Pharmacol Appl Skin Physiol 15(Suppl 1):68–73PubMedGoogle Scholar
  14. Hendrix MJ, Seftor RE, Seftor EA, Gruman LM, Lee LM, Nickoloff BJ, Miele L, Sheriff DD, Schatteman GC (2002) Transendothelial function of human metastatic melanoma cells: role of the microenvironment in cell-fate determination. Cancer Res 62:665–668PubMedGoogle Scholar
  15. Jordan SA, Jackson IJ (2000) MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles. Dev Biol 225:424–436CrossRefPubMedGoogle Scholar
  16. Kalinin A, Marekov LN, Steinert PM (2001) Assembly of the epidermal cornified cell envelope. J Cell Sci 114:3069–3070PubMedGoogle Scholar
  17. Koria P, Andreadis ST (2006) Epidermal morphogenesis: the transcriptional program of human keratinocytes during stratification. J Invest Dermatol 126:1834–1841CrossRefPubMedGoogle Scholar
  18. Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ, Carthew RW (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69–81CrossRefPubMedGoogle Scholar
  19. Lippens S, Denecker G, Ovaere P, Vandenabeele P, Declercq W (2005) Death penalty for keratinocytes: apoptosis versus cornification. Cell Death Differ 12(Suppl 2):1497–1508CrossRefPubMedGoogle Scholar
  20. Luscher-Firzlaff JM, Westendorf JM, Zwicker J, Burkhardt H, Henriksson M, Muller R, Pirollet F, Luscher B (1999) Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation. Oncogene 18:5620–5630CrossRefPubMedGoogle Scholar
  21. Mattick JS (2009) The genetic signatures of noncoding RNAs. PLoS Genet 5:e1000459CrossRefPubMedGoogle Scholar
  22. Mercer TR, Dinger ME, Sunkin SM, Mehler MF, Mattick JS (2008) Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 105:716–721CrossRefPubMedGoogle Scholar
  23. Mercer TR, Dinger ME, Mattick JS (2009) Long non-coding RNAs: insights into functions. Nat Rev Genet 10:155–159CrossRefPubMedGoogle Scholar
  24. Nickoloff BJ, Qin JZ, Chaturvedi V, Bacon P, Panella J, Denning MF (2002) Life and death signaling pathways contributing to skin cancer. J Investig Dermatol Symp Proc 7:27–35CrossRefPubMedGoogle Scholar
  25. Perera RJ, Koo S, Bennett CF, Dean NM, Gupta N, Qin JZ, Nickoloff BJ (2006a) Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways. J Cell Biochem 98:394–408CrossRefPubMedGoogle Scholar
  26. Perera RJ, Marcusson EG, Koo S, Kang X, Kim Y, White N, Dean NM (2006b) Identification of novel PPARgamma target genes in primary human adipocytes. Gene 369:90–99CrossRefPubMedGoogle Scholar
  27. Radoja N, Gazel A, Banno T, Yano S, Blumenberg M (2006) Transcriptional profiling of epidermal differentiation. Physiol Genomics 27:65–78CrossRefPubMedGoogle Scholar
  28. Seiberg M, Marthinuss J (1995) Clusterin expression within skin correlates with hair growth. Dev Dyn 202:294–301PubMedGoogle Scholar
  29. Watt FM (1989) Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol 1:1107–1115CrossRefPubMedGoogle Scholar
  30. Weijzen S, Velders MP, Elmishad AG, Bacon PE, Panella JR, Nickoloff BJ, Miele L, Kast WM (2002) The Notch ligand Jagged-1 is able to induce maturation of monocyte-derived human dendritic cells. J Immunol 169:4273–4278PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Joseph Mazar
    • 1
  • Satyabrata Sinha
    • 1
  • Marcel E. Dinger
    • 2
  • John S. Mattick
    • 2
  • Ranjan J. Perera
    • 1
    Email author
  1. 1.Sanford Burnham Medical Research InstituteOrlandoUSA
  2. 2.Institute for Molecular BioscienceUniversity of QueenslandSt. LuciaAustralia

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