Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

Abstract

Comparison of the protein expression patterns of proliferating normal primary human keratinocytes plated in serum-free medium (SFKM), supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin-binding proteins (gelsolin and tropomyosin 9220), annexins (annexins IV and VIII), hsp28, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14-3-3 family, involucrin, E-cadherin, cystatin A, desmoglein and integrins alpha 2 and beta 1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (124.0 times), MRP8 (42.4 times), MRP14 (14.9 times), tropomyosin 9220 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp28, and tropomyosin 9220 were also highly upregulated in quiescent keratinocytes indicating that their increased levels in the differentiated cells may be due to loss of proliferative activity. Highly downregulated proteins included PAI-2, tropomyosins 9213, 9121 and 9122, keratin 5, calnexin, 14-3-3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C2, alpha-enolase, eIF-4D, thioredoxin, annexins III and V, moesin, nucleolar protein B23, GST pi and PCNA/cyclin. Both the high expression of keratin 6 and 16--which are markers for an alternative pathway of keratinocyte differentiation--as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have differentiated via an abnormal pathway.

Expression of the Tumor Suppressor Protein 14-3-3σ Is Down-regulated in Invasive Transitional Cell Carcinomas of the Urinary Bladder Undergoing Epithelial-to-Mesenchymal Transition

“…5), an observation that is in line with a recent report that demonstrated that 14-3-3 expression had a tendency to be stronger in cells destined for squamous epithelium or differentiating toward a squamous cell lineage (39). High levels of 14-3-3 have also been reported in hyperproliferative skin diseases, such as psoriasis, condylomata acuminata, and actinic keratoses (40,41; see also proteomics.cancer.dk/ 2Dgallery/keratϩpso_kerat.html), suggesting again that downregulation of this protein is not an absolute requirement for cellular proliferation. However, because 14-3-3 proteins are known for their potential to regulate cellular activity by binding and sequestering proteins containing a cognate phosphorylated consensus motif (1), we cannot exclude the possibility that expression of a -interacting protein is affected in these cultured cells resulting in impaired cellular activity, functionally equivalent to down-regulation of 14-3-3.…”

Targeted ablation of Arnt in mouse epidermis results in profound defects in desquamation and epidermal barrier function

“…To assess changes in expression of epidermal genes resulting from Arnt ablation, we performed microarray hybridization on samples isolated from the epidermis of Arntnull, heterozygote with 50% of normal Arnt activity, and control mouse newborns. Unexpectedly, in Arnt ⌬/⌬ epidermis we found significant upregulation of many genes associated with advanced stages of keratinocyte terminal differentiation and formation of a functional barrier (Mischke et al, 1996;Olsen et al, 1995). This finding contradicts the accepted dogma that Arnt functions exclusively as a transcriptional activator.…”

“…Elevated levels of keratin 6 (+13.9) and keratin 16 (+3.7), which are markers of an alternative pathway of keratinocyte differentiation (Olsen et al, 1995), further confirms an abnormal terminal differentiation in the epidermis of Arnt ⌬/⌬ newborns.…”

A Combined Proteome and Ultrastructural Localization Analysis of 14-3-3 Proteins in Transformed Human Amnion (AMA) Cells

“…However, expression of 14-3-3 is also reportedly up-regulated in lung (26), head and neck (27), gastric (28), and pancreatic cancer (29,30). Additionally abnormally elevated levels of 14-3-3 have been reported in hyperproliferative skin diseases, such as psoriasis, condylomata acuminata, and actinic keratoses (31,32), showing that non-malignant human epidermal keratinocytes can express this protein at increased levels and yet display high proliferative indexes. These apparent discrepancies may stem from the different cellular roles carried out by 14-3-3 proteins in different cell types.…”