Abstract
BackgroundKeratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes.Methodology/Principal FindingsHuman keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter.Conclusions/SignificanceThe data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving PKC/AP-1 pathway, and basal-specific mediated by FOXM1, and therefore the use of K15 expression as a marker of stem cells must be viewed with caution.
Highlights
In the epidermis, the basal keratinocytes are unique due to their attachment to the basement membrane and they constitute the major proliferating cell population
We propose that there are two mechanisms regulating Keratin 15 (K15) expression in stratified epithelia; differentiation-specific involving PKC/AP-1 pathway, and basal-specific mediated by FOXM1, and the use of K15 expression as a marker of stem cells must be viewed with caution
A careful search of the K15 promoter identified a single motif (22177–2155) in this fragment, which had 75% sequence identity with the published binding motif for FOXM1B [51] (Fig. 8a). This is the first study to show that K15 is induced when keratinocytes are differentiated by anchorage deprivation in methylcellulose, or at a high cell density or by PMA
Summary
The basal keratinocytes are unique due to their attachment to the basement membrane and they constitute the major proliferating cell population. Some of the basal keratinocytes are stem cells that are characterised by their longevity and clonogenicity. These cells are responsible for tissue homeostasis and regeneration of epidermis following injury [1,2,3,4,5,6]. The committed keratinocytes downregulate integrins to become less adhesive, move to the suprabasal compartment and continue their upward movement until they are terminally differentiated [8] and shed off. This produces several layers of keratinocytes, at different stages of differentiation, which provide a protective barrier. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes
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