Shape-Induced Terminal Differentiation of Human Epidermal Stem Cells Requires p38 and Is Regulated by Histone Acetylation

John T Connelly,Julien E Gautrot,Fiona M Watt,Ajay Mishra,Laurent Kreplak

doi:10.1371/journal.pone.0027259

Abstract

Engineered model substrates are powerful tools for examining interactions between stem cells and their microenvironment. Using this approach, we have previously shown that restricted cell adhesion promotes terminal differentiation of human epidermal stem cells via activation of serum response factor (SRF) and transcription of AP-1 genes. Here we investigate the roles of p38 MAPK and histone acetylation. Inhibition of p38 activity impaired SRF transcriptional activity and shape-induced terminal differentiation of human keratinocytes. In addition, inhibiting p38 reduced histone H3 acetylation at the promoters of SRF target genes, FOS and JUNB. Although histone acetylation correlated with SRF transcriptional activity and target gene expression, treatment with the histone de-acetylase inhibitor, trichostatin A (TSA) blocked terminal differentiation on micro-patterned substrates and in suspension. TSA treatment simultaneously maintained expression of LRIG1, TP63, and ITGB1. Therefore, global histone de-acetylation represses stem cell maintenance genes independent of SRF. Our studies establish a novel role for extrinsic physical cues in the regulation of chromatin remodeling, transcription, and differentiation of human epidermal stem cells.

Highlights

Cell-extracellular matrix (ECM) interactions are key regulators of epidermal stem cell fate
Using this system to precisely control the level of adhesion and spreading of single cells, we demonstrated that limited cell adhesion promotes cortical actin polymerization and terminal differentiation in human epidermal stem cells
Inhibition of p38 mitogen activated protein kinase (MAPK) inhibited p38 mediates serum response factor (SRF) transcriptional activity Since SRF promotes keratinocyte terminal differentiation in response to restricted adhesion, we investigated whether p38 inhibition influenced SRF signaling

Summary

Introduction

Cell-extracellular matrix (ECM) interactions are key regulators of epidermal stem cell fate. We recently developed novel micro-patterned substrates on which ECM proteins are deposited in defined shapes and areas [6,7] Using this system to precisely control the level of adhesion and spreading of single cells, we demonstrated that limited cell adhesion promotes cortical actin polymerization and terminal differentiation in human epidermal stem cells. In addition to actin polymerization, SRF can be activated by the mitogen activated protein kinase (MAPK), extracellular signal-related kinase (ERK) [13] This pathway stimulates a distinct set of cofactors, which for some genes antagonize MRTF-A interactions with SRF [14,15]. Polycomb repressive complexes (PRCs) silence both senescence and terminal differentiation genes by tri-methylating lysine 27 of histone H3 (triMeK27-H3) [16] This specific mark aids chromatin packing and limits transcription [17]. Since SRF activity is sensitive to both cytoskeletal organization and MAPK signaling, we examined how various MAP kinases influence terminal differentiation using micro-patterned substrates and whether chromatin modification is involved

Results

Discussion

Findings

Materials and Methods