β-1,3/1,4-Glucan Lichenan from Cetraria islandica (L.) ACH. induces cellular differentiation of human keratinocytes

Lichenan (molecular weight 275 kDa, beta-D-1,3/1,4-glucopyranose ratio 1:3) from Cetraria islandica at a concentration of 100 mu g/mL induced terminal cellular differentiation of primary human keratinocytes as demonstrated by immunofluorescence staining using cytokeratin 10 and involucrin as marker proteins. Lichenan-derived oligosaccharides (DP3 to 8), obtained by acid-catalyzed partial hydrolysis of the polymer, did not influence cellular differentiation. Cytokeratin, filaggrin, involucrin, loricrin and transglutaminase gene expression as typical differentiation markers was increased by lichenan in a time-dependent manner. Lichenan upregulated gene cluster which were mostly related to cellular differentiation with focus on MAPK signaling as was shown by Whole Human Genome Microarray. These gene expression data from the array experiments were subsequently confirmed by qPCR for selected genes. For identification of the molecular binding structures of lichenan 1- and 2-D PAGE of keratinocyte protein membrane preparations was performed, followed by blotting with FITC-labeled lichenan and subsequent mass spectrometric identification of the pinpointed proteins. Epidermal growth factor receptor (EGFR) and integrin beta 4, both proteins being strongly involved in induction of keratinocyte differentiation were identified. In addition, protein disulfide isomerase A3 (PDIA3) showed strong binding to FITC-lichenan, indicating this enzyme to be an intracellular target of the glucan for induction of the cellular differentiation of keratinocytes. As lichenan did not influence the EGFR phosphorylation and the phosphorylation of CREB transcription factor but strongly interacted with cytosolic proteins it is hypothized that the glucan may interact with EGFR and is subsequently internalized into the cell via endosomal uptake, interacting with PDIA3, which again alters TGF beta 1 signaling towards keratinocyte differentiation.