Transcriptional signature profiling of terminal differentiation and replicative senescence in human oral keratinocytes

Abstract

Somatic cells have limited replication capacity, and after a finite number of cell divisions, DNA damage is triggered by telomere shortening

thus, the cells enter a state called replicative senescence, where DNA is unable to replicate and cell replication is irreversibly stopped. In addition, the cells that undergo replicative senescence show great differences in morphology and gene expression, even though they maintain their metabolic activity. Although there are many studies on the genes associated with differentiation and senescence, there is little information on the analysis of related genes and their gene network involved in the terminal differentiation and replicative senescence. Therefore, this study aimed to examine comprehensively the changes in the expression of the genes involved in the final differentiation and senescence process and their gene networks and functions. Isolated normal human oral keratinocytes (NHOKs) were subcultured continuously to obtain the terminally differentiated and replicative senescent cells

the state of terminal differentiation or replicative senescence of the cells was verified at the protein and RNA level. The total RNAs were isolated from rapidly growing NHOKs and replicative senescent NHOKs were analyzed by microarrays using Affymetrix Human Genome U133 Plus 2.0 Array, the results were confirmed by real-time RT-PCR, and the gene set with altered expression were run though the bioinformatical analysis software Ingenuity Pathway Analysis (IPA). Microarray results showed increased expression in 1,247 genes and reduced expression in 1,219 genes. The greatest number of these altered genes was identified as being related to biological pathways of transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response. Several highly-upregulated genes of inflammatory response (IL-1β, S100A8, S100A9, MMP1, MMP9, IL-8, BHLHB2, HES1, and TWIST1) were observed following the serial subculture in NHOKs. As the terminal differentiation and replicative senescence of NHOKs occurred, the expression of inflammation-related genes such as IL-1β and IL-8 increased. Next, in order to investigate these inflammatory genes involved in NHOKs terminal differentiation and replicative senescence, IL-1β was treated to rapidly proliferating NHOK cells for 12 to 96 hours, and terminal differentiated and replicative senescent cells were treated with IL-1β neutralizing antibody for 14 days. There was an increased IL-1β, S100A8, S100A9, MMP1, MMP9 and IL-8 expression of rapidly dividing NHOKs when treated with IL-1β, and there was a decreased gene expression of senescing NHOKs when treated with IL-1β neutralizing antibodies. Furthermore, in order to confirm this in vivo, the expressions of the genes related to the differentiation or senescence were confirmed by immunocytochemistry in gingival tissues of 3-month old and 2-year old mice. Involucrin, p16INK4A, DEC1 (Bhlhb2) and IL-1β were strongly expressed in gingival tissues of 2-year-old mice compared to that of 3-month-old mice. Overall the experiments described in this thesis presents a comprehensive analysis of the gene expression pattern of the terminal differentiation and replicative senescence process of NHOKs

these results show altered expression of genes associated with biological function of protein transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response. The purpose of this study was to lay the groundwork for the understanding of the terminal differentiation and senescence process of NHOKs by investigating the genes related to senescence through the functional analysis of gene expression changes.