Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways

Abstract

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

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

“…Previous studies (Radoja et al 2006), including our work on KC diVerentiation (Perera et al 2006a), demonstrate the involvement of a group of key regulatory genes (adhesion, ECM, proteolysis, metabolism, cell surface receptors and secreted signaling proteins, KC makers) in the KC diVerentiation pathway. Among those regulatory genes, NOTCH signaling pathway genes are known to play a major role in the regulation of diVerentiation programs in several vertebrate cell types including adipocytes (Garces et al 1997;Hendrix et al 2002;Weijzen et al 2002;Lee et al 2004).…”

“…In our previous study using monolayer cultures of KC (Perera et al 2006a), rather than EE, global gene expression proWling revealed that early conXuent KCs were somewhat similar to proliferating KCs, yet prominent diVerences were evident when compared to late conXuent KCs; which were also distinct from replicatively senescent KCs. We report here the mRNA, miRNA and long non-coding RNA diVerential gene expression proWles identiWed by using the EE culture model.…”

Notch3 Functions as a Tumor Suppressor by Controlling Cellular Senescence

“…1). Notch1 has also been shown to be down-regulated in senescent keratinocytes (50) but increased in senescent endothelial cells (51). Notch1 induces apoptosis in fibroblasts (52), neural progenitor cells (53) and B cells (54, 55), while activates senescence in endothelial cells (51) when constitutively expressed.…”

Notch tumor suppressor function

“…The role of NF-κB dependent modulation of cytokine production by Notch signaling has not been explored in great detail but it is an area of likely importance. Another major class of genes related to cytokine production that are modulated by Notch in keratinocytes are genes involved in the interferon response (Nguyen et al , 2006; Perera et al , 2006). Importantly, Notch activation in keratinocytes causes selective suppression of some interferon responsive genes, while inducing others, pointing to the existence of a novel mechanism for the fine tuning of these genes, which may be of particular significance for modulation of growth-differentiation control.…”