P16 Extracellular matrix-derived peptides regulate human keratinocyte secretory signalling
Zeyad El-Houni, Krishan Mistry, Alexander Eckersley, Sarah Herrick, Eleanor Bradley, Mike Bell, Michael SherrattAbstract
Introduction and aims
The cutaneous extracellular matrix (ECM) undergoes age-related remodelling, generating bioactive ECM-derived peptides (matrikines) with distinct signalling functions. Two in silico predicted tetrapeptide matrikines, P1 (GPKG) and P7 (LSVD), have previously been shown to modulate cultured dermal fibroblast phenotype and induce in vivo fibrillin microfibril deposition at the dermoepidermal junction (DEJ). However, their effects on epidermal function remain poorly defined. Recently, we have shown that cultured keratinocytes (primary cells from multiple donors) respond more rapidly and robustly to these peptides than dermal fibroblasts, exhibiting increased proliferation, extensive transcriptional reprogramming, and accelerated re-epithelialization in a three-dimensional wound-healing model (immortalized keratinocyte cell line). Here, we sought to determine whether peptide treatment affects the keratinocyte secretome, which mediates autocrine and paracrine regulation of keratinocyte behaviour, inflammation, barrier and DEJ repair.
Methods
Pooled primary human epidermal keratinocytes from two donors (PromoCell) were treated with P1, P7, P1+P7, or vehicle control. Conditioned media (n = 5) was collected at 8, 24 and 48 h post-treatment and analysed by data-independent acquisition (DIA) mass spectrometry (DIA-MS) on the Astral LC-MS/MS platform. For differential expression analysis using DIA neural networks, proteins were filtered based on subcellular localization, retaining secreted or extracellular molecules.
Results
Across all conditions, DIA-MS identified approximately 1450 differentially abundant secreted proteins (log2 fold change ≥ 0.58; q < 0.05). Secretome-restricted pathway analysis revealed distinct responses to peptide treatment. P1 and P7 administered separately elicited early wound- and cytokine-associated paracrine pathways that resolved over time. In contrast, the P1+P7 combination preferentially activated lipid/nuclear-receptor synthesis while suppressing mediators of inflammatory and fibrotic signalling, consistent with a homeostatic, barrier-supportive secretory phenotype. Notably, P1+P7 significantly induced COL7A1 deposition, a key mediator of DEJ adhesion.
Conclusions
These findings show that matrikine peptides profoundly remodel the keratinocyte secretome, revealing a previously underappreciated mechanism by which ECM-derived signals regulate epidermal–dermal crosstalk and skin homeostasis.