Two Mechanisms Regulate Keratin K15 Expression In Keratinocytes: Role of PKC/AP-1 and FOXM1 Mediated Signalling

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.

The Protein Deacetylase SIRT3 Prevents Oxidative Stress-induced Keratinocyte Differentiation

“…Previous studies suggest that ROS signaling might induce keratinocyte differentiation through the PKC/AP-1 pathway (24,25). Interestingly, SIRT3 gene expression might in turn also be regulated through AP-1 transcription factors (26), suggesting a possible regulatory pathway.…”

Construction and characterization of a multilayered gingival keratinocyte culture model: the TURK-U model

“…When fibronectin was replaced with laminin-332, the cells produced multilayers but did not attach to the membrane. It has been reported that methylcellulose (1.13 %) induced irreversible loss of keratinocyte proliferation but the effect could be reversed by fibronectin (Bose et al 2012). This finding may explain the dependence of our model on fibronectin.…”

Exploiting FOXM1‐orchestrated molecular network for early squamous cell carcinoma diagnosis and prognosis

“…By exploiting the aberrant expression of FOXM1 found in many human cancers,38 we have developed a novel molecular diagnostic system that quantify the levels of cell proliferation,38 differentiation,10 ageing,40 genomic instability,2, 14, 16, 17, 19 epigenetic11, 14 and stem cell reprogramming10, 13, 41, 42 as a collective basis for cancer diagnosis. Our proof‐of‐principal studies tested qMIDS on multiple cohorts of patients with HNSCC and demonstrated that the qMIDS assay is a practical, sensitive, objective, quantitative and customisable method for cancer diagnosis.…”

“…FOXM1 cell cycle transcription factor is involved in cancer initiation,10–13 progression14–16 and metastasis,10, 17–19 and ultimately drug resistance 12, 20–23. Here, we exploited the value of FOXM1 (isoform B)‐orchestrated transcriptional changes in 14 target genes and 2 reference genes as a ‘molecular gauge’ to quantify malignancy status of tissue specimens using a highly sensitive absolute quantitative reverse transcription PCR (qPCR) method and demonstrate a prototype multibiomarker ‘quantitative molecular index diagnostic system’ (qMIDS) for squamous cell carcinoma (SCC) detection.…”