γ-Propoxy-Sulfo-Lichenan Induces In Vitro Cell Differentiation of Human Keratinocytes

Abstract

Background: As non-cellulosic β-d-glucans are known to exert wound-healing activity by triggering keratinocytes into cellular differentiation, the functionality of a semisynthetic lichenan-based polysaccharide on skin cell physiology was investigated.

Methods: γ-Propoxy-sulfo-lichenan (γ-PSL, molecular weight 52 kDa, β-1,3/1,4-p-d-Glucose, degree of substitution 0.7) was prepared from lichenan. Differentiation of primary human keratinocytes was assayed by the protein analysis of differentiation specific markers and by gene expression analysis (qPCR). The gene array gave insight into the cell signaling induced by the polysaccharide.

Results: γ-PSL (1 to 100 μg/mL) triggered keratinocytes, in a concentration-dependent manner, into the terminal differentiation, as shown by the increased protein expression of cytokeratin 1 (KRT1). Time-dependent gene expression analysis proved differentiation-inducing effects, indicating strong and fast KRT1 gene expression, while KRT10 expression showed a maximum after 12 to 24 h, followed by downregulation to the basal level. Involucrin gene expression was only changed to a minor extent, which was similar to loricrin and transglutaminase. Gene array indicated the influence of γ-PSL on MAP kinase and TGF-β mediated signaling towards keratinocyte differentiation.

Conclusion: The propoxylated lichenan may improve wound healing by topical application to promote the terminal barrier formation of keratinocytes.

Keywords: differentiation; involucrin; keratin; keratinocytes; lichenan; γ-propoxy-sulfo-lichenan.

Figure 1

Synthesis of γ-propoxy-sulfo-lichenan (PSL) from lichenan and γ-propan sulton under alkaline conditions.

Figure 2

Relevant details of the ¹H-¹³C-HSQC-NMR spectrum of γ-PSL. Additional signals of γ-PSL, in comparison to unsubstituted lichenan, are marked by arrows.

Figure 3

Influence of γ-PSL (1 to 100 μg/mL) on the expression of differentiation-specific marker proteins KRT1, KRT10 (A), and IVL (B) in natural human epidermal keratinocytes (NHEK) after 7 days of incubation. Loading control: β-actin (10 µg protein/sample); positive controls: Ca²⁺ 1.8 mM; DC: Double cell density, contact inhibition; and UC: Untreated control. Note: KRT1 antibody partly produced artifacts.

Figure 4

Influence of γ-PSL (100 µg/mL) on the relative, normalized gene expression of differentiation specific marker genes in NHEKs by time dependent qPCR over 12–60 h. FLG: filaggrin, IVL: Involucrin, KRT1: Cytokeratin 1, KRT10: Cytokeratin 10, LOR: Loricrin, and TGM1: Transglutaminase 1. Relative, normalized expression, related to the untreated control (expression = 1).

Figure 5

Quantitative Real-Time Array (Development-Keratinocyte differentiation H96 array—BioRad) after 60 h incubation of NHEK with γ-PSL (100 µg/mL) on the relative, normalized gene expression of differentiation specific genes in relation to the untreated control (normalized expression = 1); #: genes selected for subsequent qPCR cross validation. Data are related to the independent array experiments (A,B).

Figure 5

Quantitative Real-Time Array (Development-Keratinocyte differentiation H96 array—BioRad) after 60 h incubation of NHEK with γ-PSL (100 µg/mL) on the relative, normalized gene expression of differentiation specific genes in relation to the untreated control (normalized expression = 1); #: genes selected for subsequent qPCR cross validation. Data are related to the independent array experiments (A,B).

Figure 6

Potential mode of action of γ-PSL on NHEK, by interfering with the interaction of integrins on the cell surface with proliferation signals from the extracellular matrix.