Effects of streptolysin o on extracellular matrix gene expression in normal human epidermal keratinocytes

Abstract

ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05's mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hours, followed by RNA extraction and cRNA production. Gene expression profiling utilized microarrays containing nucleic acid probes for 113 extracellular matrix (ECM) genes. Microarrays yielded 6 upregulated and 4 downregulated genes with >2-fold changes and p<0.05 in t-tests. Quantitative real-time polymerase chain reactions (qPCR) were used to verify gene regulation. Upregulated genes of interest were VCAN (formerly CSPG2, encoding versican), CD44 (encoding hyaluronan receptor), ICAM1 (encoding intercellular adhesion molecule-1) and CTGF (encoding connective tissue growth factor). All four upregulated genes encode proteins involved in promoting keratinocyte migration and proliferation. Downregulated genes of interest were MMP9 (encoding matrix metallopro-teinase 9) and SPP1 (encoding osteopontin). ML-05 may enhance wound healing through the expression of specific genes encoding proteins capable of promoting keratinocyte migration, proliferation, and other activities related to maintaining ECM structure and function ©2011 University of Massachusetts.