As the keratinocyte differentiation process occurs along a pathway that leads to cell cycle arrest and terminal differentiation, a complex program of gene expression must be coordinated1,8
. Many differentiation-related genes, including those encoding transglutaminases 1 and 3, involucrin, cornifin, loricrin, filaggrin, and small proline-rich proteins, are expressed in a temporally regulated manner9,10
. New genes such as Brn211
, Nkx 2.512
, plasminogen activator inhibitor-213
has been suggested to play a role in keratinocyte differentiation.
There are three major molecular mechanisms mediating epigenetic change. They are DNA methylation, Histone modification, and MicroRNA interference4
. Methylation of the cytosine and guanine dinucleotides (CpG islands) occurs in the promoter region of approximately 40% of genes in higher eukaryotes. Methylation usually represses gene transcription14
, While histone acetylation is generally linked to activation of transcription15
. Another mechanism is the effect of nonprotein coding mRNAs on the transcription of other genes and protein synthesis16
. The microRNA pathways may be more promising as therapeutic targets, as their effects are more specific than methylation or histone modification.
Knowledge of the contribution of epigenetic mechanisms to the pathogenesis of skin disease has expanded considerably over the last few years, particularly in the field of skin cancer and inflammatory skin diseases9,17
. Despite intensive research into skin disease, epigenetic modulation during keratinocyte differentiation is not yet understood.
We used trypsin for epidermal fragmentation, as described previously18
with successive short-term enzyme incubation to progressively detach cells from the deep layers, and to purify the cells. Incubations were performed at 4
to stop cellular metabolic activity and to preserve the mRNA pool from degradation. This point is crucial as many growth factors, cell cycle regulators, and transcription factors are encoded by short-lived mRNAs.
We performed methylation DNA microarray analysis with genomic DNA isolated from the basal (T1) and cornified layers (T4). A Methylated-CpG assisted microarray analysis was performed as described previously19
. We examined 9 hypermethylated genes and 9 hypomethylated genes. Most of these genes had not previously been associated with keratinocyte differentiation. PSCD2 (pleckstrin homology, Sec7 and coiled-coil domain 2) was the most significant gene in hypermethylation field, and YWHAQ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide) was the most reliable gene in hypomethylation field.
The PSCD2 gene functions to promote activation of adenosin diphosphate-ribosylation factor (ARF) through replacement of GDP with GTP. Members of this family have an identical structural organization that consists of an N-terminal coiled-coil motif, a central Sec7 domain, and a C-terminal pleckstrin homology domain20
. The functions of this family include mediating regulation of protein sorting and membrane trafficking20,21
. Although there are reports of a regulatory role in development of neuronal processes22
, the role of PSCD2 during keratinocyte differentiation has not yet been investigated.
YWHAQ is a gene associated with an adapter protein that is implicated in regulation of a signaling pathway. Binding generally results in modulation of the activity of the binding partner (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide)23,24
. YWHAQ is directly involved in cellular processes, such as cytokinesis, cell-contact inhibition, anchorage-independent growth, and cell adhesion, processes that often become deregulated in diseases like cancer24,25
. Recently, there was a report that YWHAZ, an isoform of YWHAQ, significantly suppressed the growth rate of head and neck squamous cell carcinoma cell lines, and overexpression of YWHAZ in human keratinocytes promotes overgrowth and morphological changes26,27
We have identified many hypermethylated and hypomethylated genes from differentiated keratinocytes that are involved in epigenetic regulation of keratinocyte differentiation. As this is a preliminary study, more work is necessary to determine whether changes in the methylation status of these candidate genes actually control keratinocyte differentiation. Although extensive work in this field is clearly needed, our preliminary findings highlight the importance of epigenetic modulation in keratinocyte differentiation-specific gene regulation. Furthermore, we provide useful information for future development of novel therapeutic and preventive tools for many skin diseases associated with abnormalities in keratinocyte differentiation.